Methods and unit dose formulations for the inhalation administration of tobramycin

ABSTRACT

A patient suffering from an endobronchial infection is treated by administering to the patient for inhalation a dose of less than about 4.0 ml of a nebulized aerosol formulation comprising from about 60 to about 200 mg/ml of an aminoglycoside antibiotic, such as tobramycin, in a physiologically acceptable carrier in a time period of less than about 10 minutes. Unit dose devices for storage and delivery of the aminoglycoside antibiotic formulations are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/151,701, filed May 17, 2002 now abandoned, which claims the benefitof U.S. Provisional Application No. 60/292,234, filed May 18, 2001.

FIELD OF THE INVENTION

The present invention relates to new and improved unit dose containersof aminoglycoside antibiotics, such as tobramycin, for delivery byaerosol inhalation, and to improved methods of treatment of susceptibleacute or chronic endobronchial infections.

BACKGROUND OF THE INVENTION

Progressive pulmonary disease is the cause of death in over 90% ofcystic fibrosis (CF) patients (Koch C. et al., “Pathogenesis of cysticfibrosis,” Lancet 341(8852):1065-9 (1993); Konstan M. W. et al.,“Infection and inflammation of the lung in cystic fibrosis,” Davis P B,ed., Lung Biology in Health and Disease, Vol. 64. New York, NY: Dekker:219-76 (1993)). Pseudomonas aeruginosa is the most significant pathogenin CF lung disease. Over 80% of CF patients eventually become colonizedwith P. aeruginosa (Fitzsimmons S. C., “The changing epidemiology ofcystic fibrosis,” J Pediatr 122(1):1-9 (1993)). The standard therapy forP. aeruginosa endobronchial infections is 14 to 21 days of parenteralantipseudomonal antibiotics, typically including an aminoglycoside.However, parenteral aminoglycosides, as highly polar agents, penetratepoorly into the endobronchial space. To obtain adequate drugconcentrations at the site of infection with parenteral administration,serum levels approaching those associated with nephro-, vestibule-, andoto-toxicity are required (“American Academy of Otolaryngology. Guidefor the evaluation of hearing handicap,” JAMA 241(19):2055-9 (1979);Brummett R. E., “Drug-induced ototoxicity,” Drugs 19:412-28 (1980)).

Aerosolized administration of aminoglycosides offers an attractivealternative, delivering high concentrations of antibiotic directly tothe site of infection in the endobronchial space while minimizingsystemic bioavailability (Touw D. J. et al., “Inhalation of antibioticsin cystic fibrosis,” Eur Respir J 8:1594-604 (1995); Rosenfeld M. etal., “Aerosolized antibiotics for bacterial lower airway infections:principles, efficacy, and pitfalls,” Clinical Pulmonary Medicine4(2):101-12(1997)).

Tobramycin is commonly prescribed for the treatment of serious P.aeruginosa infections. It is an aminoglycoside antibiotic produced bythe actinomycete, Streptomyces tenebrarius. Low concentrations oftobramycin (<4 μg/mL) are effective in inhibiting the growth of manyGram-negative bacteria and under certain conditions may be bactericidal(Neu, H. C., “Tobramycin: an overview,” J Infect Dis 134, Suppl: S3-19(1976)). Tobramycin is poorly absorbed across mucosal surfaces,conventionally necessitating parenteral administration. Tobramycinactivity is inhibited by purulent sputum: high concentrations ofdivalent cations, acidic conditions, increased ionic strength andmacromolecules that bind the drug all inhibit tobramycin in thisenvironment. It is estimated that 5 to 10 times higher concentrations oftobramycin are required in the sputum to overcome these inhibitoryeffects (Levy J. et al., “Bioactivity of gentamicin in purulent sputumfrom patients with cystic fibrosis or bronchiectasis: comparison withactivity in serum,” J Infect Dis 148(6):1069-76 (1983)).

Delivery of the poorly absorbed antibiotic tobramycin to the airway bythe aerosol route of cystic fibrosis (CF) patients has been documentedusing the aerosol route. Much of this work has been done towardtreatment of chronic lung infections with P. aeruginosa in cysticfibrosis (CF) patients. A multicenter, double blind, placebo-controlled,crossover trial of 600 mg tid of aerosolized tobramycin forendobronchial infections due to P. aeruginosa in 71 CF patientsdemonstrated a significant reduction in sputum density of this pathogenas well as improved spirometry in the treatment group. Emergence of P.aeruginosa strains highly resistant to tobramycin (defined as MIC≧128μg/mL) was comparable in the placebo and treatment groups. The presencein the sputum of Gram-negative organisms other than P. aeruginosaintrinsically resistant to tobramycin occurred with equal frequencyduring administration of tobramycin or placebo (Ramsey B. et al.,“Response to Letter to the Editor: Aerosolized tobramycin in patientswith cystic fibrosis,” N Engl J Med 329:1660 (1993)).

Although this regimen was found to be both safe and efficacious, it iscostly and inconvenient. A survey of the MICs for P. aeruginosa isolatesfrom initial sputum cultures for patients at the Children's Hospital CFCenter, Seattle, Wash., in 1993 found that 90% of isolates had MICs≦16μg/mL and 98% of all isolates had MICs≦128 μg/mL. This survey suggestedthat achieving a sputum tobramycin concentration of 128 μg/mL shouldtreat the endobronchial infection in CF patients (Levy J. et al.,“Bioactivity of gentamicin in purulent sputum from patients with cysticfibrosis or bronchiectasis: comparison with activity in serum,” J InfectDis 148(6):1069-76 (1983)).

A randomized, crossover study compared the ability of several nebulizersto deliver tobramycin by measuring peak sputum tobramycin concentrationsin samples collected ten minutes after completion of the aerosol dose.This study administered TOBI® tobramycin solution for inhalation,PathoGenesis Corporation, Seattle, Wash. (now Chiron Corporation,Emeryville, Calif.), containing 60 mg/mL tobramycin in 5 mL one quarter(¼) normal saline, using the Pari® LC jet nebulizer, Pari RespiratoryEquipment, Inc., Richmond, Va. This delivery system was shown to delivera mean peak sputum tobramycin concentration of 678.8 μg/g (s.d. 661.0μg/g), and a median peak sputum concentration of 433.0 μg/g. Only 13% ofpatients had sputum levels ≦128 μg/g; 87% of patients achieved sputumlevels of ≧128 μg/g (Eisenberg, J. et al., “A Comparison of Peak SputumTobramycin Concentration in Patients With Cystic Fibrosis Using Jet andUltrasonic Nebulizer Systems. Aerosolized Tobramycin Study Group,” Chest111(4):955-962 (1997)). Recently, the Pari® LC jet nebulizer has beenmodified with the addition of one-way flow valves, and renamed the Pari®LC PLUS. The one-way valves in the Pari® LC PLUS have been described aspermitting the delivery of more drug than the Pari® LC jet nebulizer,while decreasing the potential for accidental spillage and allowing forthe use of an expiratory filter. Experience has shown that mean peaksputum tobramycin concentrations achieved using the Pari LC PLUS jetnebulizer are significantly higher than those using the Pari® LC jetnebulizer as described in Eisenberg et al. (1997), supra.

Two placebo-controlled, multicenter, randomized, double blind clinicaltrials of intermittent administration of inhaled tobramycin in cysticfibrosis patients with P. aeruginosa infection were reported in Ramsey,B. W. et al., “Intermittent Administration of Inhaled Tobramycin inPatients with Cystic Fibrosis. Cystic Fibrosis Inhaled Tobramycin StudyGroup.” N. Engl. J. Med. 340(1):23-30 (1999). In these studies, fivehundred twenty subjects were randomized to receive either 300 mg inhaledtobramycin or placebo twice daily for 28 days followed by 28 days offstudy drug. Subjects continued on treatment or placebo for three“on-off” cycles for a total of 24 weeks. Efficacy variables includedsputum P. aeruginosa density. Tobramycin-treated patients had an average0.8 log₁₀ decrease in P. aeruginosa density from Week 0 to Week 20,compared with a 0.3 log₁₀ increase in placebo-treated patients(P<0.001). Tobramycin-treated patients had an average 1.9 log₁₀ decreasein P. aeruginosa density from Week 0 to Week 4, compared with no changein placebo-treated patients (P<0.001).

A preservative-free, stable, and convenient formulation of tobramycin(TOBI® tobramycin solution for inhalation; 60 mg/mL tobramycin in 5 mLof ¼ normal saline) for administration via jet nebulizer was developedby PathoGenesis Corporation, Seattle, Wash. (now Chiron Corporation,Emeryville, Calif.). The combination of a 5 mL BID TOBI dose (300 mgtobramycin) and the PARI LC PLUS/PulmoAide compressor delivery systemwas approved under NDA 50-753, December 1997, for the management of P.aeruginosa in CF patients, and remains the industry standard for thispurpose. The aerosol administration of a 5 ml dose of a formulationcontaining 300 mg of tobramycin in quarter normal saline for thesuppression of P. aeruginosa in the endobronchial space of a patient isdisclosed in U.S. Pat. No. 5,508,269, the disclosure of which isincorporated herein in its entirety by this reference.

Although the current conventional delivery systems have been shown to beclinically efficacious, they typically suffer from relatively lowefficiency levels in delivering antibiotic solutions to theendobronchial space of a patient, thereby wasting a substantial portionof the nebulized antibiotic formulations and substantially increasingdrug delivery costs. The low efficiency of current conventional deliverysystems requires patients to devote relatively long time periods toreceive an effective dose of the nebulized antibiotic formulations,which can lead to decreased patient compliance. Accordingly, there is aneed for new and improved methods and devices for the delivery ofaminoglycoside antibiotic compounds to a patient by inhalation to reduceadministration costs, increase patient compliance and enhance overalleffectiveness of the inhalation therapy.

SUMMARY OF THE INVENTION

It has now been discovered that patients suffering from an endobronchialinfection can be effectively and efficiently treated by administering tothe patient for inhalation a dose of less than about 4.0 ml of anebulized aerosol formulation comprising from about 60 to about 200mg/ml of an aminoglycoside antibiotic, such as tobramycin, in aphysiologically acceptable carrier in a time period of less than about10 minutes, more preferably less than about 8 minutes, and even morepreferably less than about 6 minutes. In other aspects, the administereddose may be less than about 3.75 ml or 3.5 ml or less, and theaminoglycoside antibiotic formulation may comprise from about 80 toabout 180 mg/ml of aminoglycoside antibiotic or more preferably fromabout 90 to about 150 mg/ml of aminoglycoside antibiotic.

In other aspects, the present invention provides unit dose formulationsand devices adapted for use in connection with a high efficiencyinhalation system, the unit dose device comprising a container designedto hold and store the relatively small volumes of the aminoglycosideantibiotic formulations of the invention, and to deliver theformulations to an inhalation device for delivery to a patient inaerosol form. In one aspect, a unit dose device of the inventioncomprises a sealed container, such as an ampoule, containing less thanabout 4.0 ml of an aminoglycoside antibiotic formulation comprising fromabout 60 to about 200 mg/ml of an aminoglycoside antibiotic in aphysiologically acceptable carrier. The sealed container is preferablyadapted to deliver the aminoglycoside antibiotic formulation to a highefficiency inhalation device for aerosolization and inhalation by apatient. In other aspects, the container of the unit dose device maycontain less than about 3.75 ml, or 3.5 ml or less, of theaminoglycoside antibiotic formulation, and the aminoglycoside antibioticformulation may comprise from about 80 to about 180 mg/ml, or from about90 to about 120 mg/ml, of aminoglycoside antibiotic.

In yet other aspects, the present invention relates to a system fordelivering an aminoglycoside antibiotic formulation to a patient in needof such treatment, comprising a unit dose device as described in detailabove, comprising a container containing less than about 4.0 ml of anaminoglycoside antibiotic formulation comprising from about 60 to about200 mg/ml of an aminoglycoside antibiotic in a physiologicallyacceptable carrier, and means for delivering the aminoglycosideantibiotic formulation from the unit dose device for inhalation by thepatient in aerosolized form in less that 10 about minutes.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 is a graphical representation illustrating the mean relativechanges in FEV₁% predicted from before to 30 minutes after dosing with300 mg tobramycin with a PARI LC PLUS jet nebulizer/PulmoAide compressordelivery system, or with 30, 60, or 90 mg tobramycin with an Aerodosebreath actuated nebulizer, as described in Example 1;

FIG. 2 is a graphical representation showing sputum tobramycinconcentrations by time from dosing by the tobramycin formulations ofFIG. 1, as described in Example 1;

FIG. 3 is a graphical representation showing sputum maximum plasmaconcentrations (C_(max)) following dosing by the tobramycin formulationsof FIG. 1, as described in Example 1;

FIG. 4 is a graphical representation showing sputum area under theplasma concentration time profile (AUC₀₋₈) following dosing by thetobramycin formulations of FIG. 1, as described in Example 1;

FIG. 5 is a graphical representation showing serum tobramycinconcentrations by time following dosing by the tobramycin formulationsof FIG. 1, as described in Example 1;

FIG. 6 is a graphical representation showing serum maximum plasmaconcentrations (C_(max)) following dosing by the tobramycin formulationsof FIG. 1, as described in Example 1;

FIG. 7 is a graphical representation showing serum area under the plasmaconcentration time profile (AUC₀₋₈) following dosing by the tobramycinformulations of FIG. 1, as described in Example 1;

FIG. 8 is a graphical representation showing the mean recovery oftobramycin from urine 0-8, 8-24 and 0-24 hours post dosing with theformulations of FIG. 1, as described in Example 1; and

FIG. 9 is a graphical representation showing the mean nebulization timein minutes for dosing with the formulations of FIG. 1, as described inExample 1.

FIG. 10 is a graphical representation showing the average serum-timeprofiles of tobramycin after administration of 300 mg tobramycin (TOBI)and 420 mg tobramycin solution for inhalation (TSI), as described inExample 3.

FIG. 11 is a graphical representation showing the average sputum-timeprofiles of tobramycin after administration of 300 mg tobramycin (TOBI)and 420 mg tobramycin solution for inhalation (TSI), as described inExample 3.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In accordance with the present invention, methods are provided for thetreatment of a patient in need of treatment, such as a patient sufferingfrom an endobronchial P. aeruginosa infection, comprising administeringto the patient for inhalation a relatively small volume of anaminoglycoside antibiotic formulation over a relatively short period oftime. This aspect of the invention is particularly suitable forformulation of concentrated aminoglycosides, such as tobramycin, foraerosolization by small volume, breath actuated, high output rate andhigh efficiency inhalers to produce a aminoglycoside aerosol particlesize between 1 and 5 μm desirable for efficacious delivery of theaminoglycoside into the endobronchial space to treat susceptiblemicrobial infections, such as Pseudomonas aeruginosa infections. Theformulations preferably contains minimal yet efficacious amount ofaminoglycoside formulated in smallest practical volume of aphysiologically acceptable solution, for example an aqueous solutionhaving a salinity adjusted to permit generation of aminoglycosideaerosol particles that are well-tolerated by patients but preventing thedevelopment of secondary undesirable side effects such as bronchospasmand cough. By the more efficient administration of the aminoglycosideformulation provided by the present invention, substantially smallervolumes of aminoglycoside than the conventional administration regimeare administered in substantially shorter periods of time, therebyreducing the costs of administration and drug waste, and significantlyenhancing the likelihood of patient compliance.

Thus, in accordance with one aspect of the present invention, methodsare provided for the treatment of a patient in need of treatment, suchas a patient suffering from an endobronchial P. aeruginosa infection,comprising administering to the patient for inhalation a dose of lessthan about 4.0 ml of a nebulized aerosol formulation comprising fromabout 60 to about 200 mg/ml of an aminoglycoside antibiotic in a timeperiod of less than about 10 minutes. In other aspects, the dose of theaerosol formulation is administered to the patient in less than about 8minutes. In yet other aspects, the dose of the aerosol formulation isadministered to the patient in less than about 6 minutes.

The aerosol formulations administered in the practice of the inventionmay comprise from about 60 to about 200 mg/ml of aminoglycosideantibiotic. In other aspects of the invention, the aerosol formulationsadministered in the practice of the invention may comprise from about 80to about 180 mg/ml of aminoglycoside antibiotic. In yet other aspects ofthe invention, the aerosol formulations administered in the practice ofthe invention may comprise from about 90 to about 150 mg/ml ofaminoglycoside antibiotic.

In the practice of the methods of the invention, substantially smallervolumes of aerosol formulation are administered to the patient, ascompared with the conventional administration processes. Thus, in oneaspect a dose of less than about 4.0 ml of a nebulized aerosolformulation is administered to the patient. In another aspect, a dose ofless than about 3.75 ml of a nebulized aerosol formulation isadministered to the patient. In yet another aspect, a dose of 3.5 ml orless of a nebulized aerosol formulation is administered to the patient.

In yet other aspects, the present invention relates to a system fordelivering an aminoglycoside antibiotic formulation to a patient in needof such treatment, comprising a unit dose device as described in detailherein, comprising a container containing less than about 4.0 ml of anaminoglycoside antibiotic formulation comprising from about 60 to about200 mg/ml of an aminoglycoside antibiotic in a physiologicallyacceptable carrier, and means for delivering the aminoglycosideantibiotic formulation from the unit dose device for inhalation by thepatient in aerosolized form in less that 10 about minutes.

In order to deliver the relatively small volumes of the relatively highconcentration aminoglycoside antibiotic formulations to the patient forinhalation in the relatively short dosing periods of the invention, theantibiotic formulations are preferably administered with the use of aninhalation device having a relatively high rate of aerosol output.Useful devices may also exhibit high emitted dose efficiency (i.e., lowresidual volume in the device). In order to increase the overallefficiency of the system, emission may additionally be limited toperiods of actual inhalation by the patient (i.e., breath actuated).Thus, while conventional air-jet nebulizers exhibit a rate of aerosoloutput on the order of 3 μl/sec, inhalation devices useful for use inthe practice of the present invention will typically exhibit a rate ofaerosol output of not less that about 4 μl/sec. In some cases,inhalation devices useful for use in the practice of the presentinvention will exhibit a rate of aerosol output of not less than about 5μl/sec or even not less than about 8 μl/sec. In addition, whileconventional air-jet nebulizers have a relatively low emitted doseefficiency and typically release about 55% (or less) of the nominal doseas aerosol, inhalation devices useful for use in the practice of thepresent invention may release at least about 75%, more preferably atleast about 80% and most preferably at least about 85% of the loadeddose as aerosol for inhalation by the patient. In other aspects,conventional air-jet nebulizers typically continually releaseaerosolized drug throughout the delivery period, without regard towhether the patient is inhaling, exhaling or in a static portion of thebreathing cycle, thereby wasting a substantial portion of the loadeddrug dose. In some embodiments, inhalation devices for use in thepresent invention will be breath actuated, and restricted to delivery ofaerosolized particles of the aminoglycoside formulation to the period ofactual inhalation by the patient. One representative inhalation devicemeeting the above criteria and suitable for use in the practice of theinvention is the Aerodose™ inhaler, available from Aerogen, Inc.,Sunnyvale, Calif. The Aerodose™ inhaler generates an aerosol using aporous membrane driven by a piezoelectric oscillator. Aerosol deliveryis breath actuated, and restricted to the inhalation phase of the breathcycle, i.e., aerosolization does not occur during the exhalation phaseof the breath cycle. The airflow path design allows normal inhale-exhalebreathing, compared to breath-hold inhalers. Additionally, the Aerodose™inhaler is a hand-held, self-contained, and easily transported inhaler.Although piezoelectric oscillator aerosol generators, such as theAerodose™ inhaler, represent one embodiment for use in the practice ofthe invention, other inhaler or nebulizer devices may be employed thatmeet the above performance criteria and are capable of delivering thesmall dosage volumes of the invention with a relative high effectivedeposition rate in a comparatively short period of time. In otherembodiments of the invention devices useful for delivering theconcentrated aminoglycoside formulations of the invention includeconventional air-jet nebulizers coupled with a compressor capable ofhigher than conventional output pressures. Enhanced compressor outputpressures useful in the practice of the invention will be readilydeterminable to those skilled in the art in view of the disclosurecontained herein. As one representative example, the PARI LC PLUS™ jetnebulizer, PARI GmbH, Stamberg, Germany, driven by a Invacare MOBILAIRE™compressor, Invacare Corporation, Elyria, Ohio, set for an outputpressure of about 35 psi has been found to be capable of delivering 3.5ml of the concentrated aerosolized aminoglycoside formulations of theinvention (such as tobramycin) in 10 minutes or less, as is hereinafterdescribed in detail in Example 3.

Aminoglycoside antibiotics useful in the practice of the inventioninclude, for example, gentamicin, amikacin, kanamycin, streptomycin,neomycin, netilmicin and tobramycin. A presently particularly preferredaminoglycoside antibiotic for this purpose is tobramycin. Formulationsaccording to the invention typically contain from about 60 to about 200mg, more preferably from about 80 to about 180, and most preferably fromabout 90 to about 120 mg of aminoglycoside per ml of solution. Theaminoglycoside antibiotic of the invention may be incorporated intosterile water or physiologically acceptable solution. Other componentsmay be included in the formulation, as desired. In order to facilitateadministration and compatibility with the endobronchial space, theaminoglycoside antibiotic of the invention is preferably formulated in adiluted physiological saline solution, such as in one quarter strengthof normal saline, having a salinity adjusted to permit generation oftobramycin aerosol well-tolerated by patients but to prevent thedevelopment of secondary undesirable side effects such as bronchospasmand cough. Typically, about 90 to about 120 mg of aminoglycosideantibiotic is dissolved in 1 ml solution of a diluted, typically quarternormal saline containing about 0.225% NaCl. Quarter normal saline, thatis 0.225% of sodium chloride, is a presently preferred vehicle fordelivery of aminoglycoside into endobronchial space.

By way of illustration, high concentrations of tobramycin administeredto the lungs by aerosolization result in maximization of sputum levelsof tobramycin and in minimization of tobramycin serum levels. Thus,administration of tobramycin by aerosolization has the advantage ofreducing systemic toxicity while providing efficacious concentrations oftobramycin in the sputum. The bronchial barrier restricts the movementof aerosolized tobramycin and prevents it from reaching high systemiclevels.

In other aspects of the present invention, unit dose formulations anddevices are provided for administration of an aminoglycoside antibioticformulation to a patient with an inhaler, in accordance with the methodsof the invention as described supra. Preferred unit dose devicescomprise a container designed to hold and store the relatively smallvolumes of the aminoglycoside antibiotic formulations of the invention,and to deliver the formulations to an inhalation device for delivery toa patient in aerosol form. In one aspect, unit dose containers of theinvention comprise a plastic ampoule filled with an aminoglycosideantibiotic formulation of the invention, and sealed under sterileconditions. Preferably, the unit dose ampoule is provided with atwist-off tab or other easy opening device for opening of the ampouleand delivery of the aminoglycoside antibiotic formulation to theinhalation device. Ampoules for containing drug formulations are wellknown to those skilled in the art (see, for example, U.S. Pat. Nos.5,409,125, 5,379,898, 5,213,860, 5,046,627, 4,995,519, 4,979,630,4,951,822, 4,502,616 and 3,993,223, the disclosures of which areincorporated herein by this reference). The unit dose containers of theinvention may be designed to be inserted directly into an inhalationdevice of the invention for delivery of the contained aminoglycosideantibiotic formulation to the inhalation device and ultimately to thepatient.

In accordance with this aspect of the invention, a unit dose device isprovided comprising a sealed container containing less than about 4.0 mlof an aminoglycoside antibiotic formulation comprising from about 60 toabout 200 mg/ml of an aminoglycoside antibiotic in a physiologicallyacceptable carrier, the sealed container being adapted to deliver theaminoglycoside antibiotic formulation to an inhalation device foraerosolization. Suitable aminoglycoside antibiotics for use inconnection with this aspect of the invention include thoseaminoglycoside antibiotics described in detail, supra. In a presentlypreferred embodiment, the aminoglycoside antibiotic employed in the unitdose devices of the invention is tobramycin. In other aspects, the unitdose devices of the invention contain less than about 3.75 ml of theaminoglycoside solution. In other aspects, the unit dose devices of theinvention contain 3.5 ml or less of the aminoglycoside solution.

In other aspects of the invention, the unit dose devices of theinvention may contain an aminoglycoside antibiotic formulationcomprising from about 80 to about 180 mg/ml of aminoglycosideantibiotic. In yet other aspects of the invention, the unit dose devicesof the invention may contain an aminoglycoside antibiotic formulationcomprising from about 90 to about 150 mg/ml of aminoglycosideantibiotic.

In preferred unit dose formulations of the invention, thephysiologically acceptable carrier may comprise a physiological salinesolution, such as a solution of one quarter strength of normal saline,having a salinity adjusted to permit generation of a tobramycin aerosolthat is well-tolerated by patients, but that prevents the development ofsecondary undesirable side effects such as bronchospasm and cough.

These and other aspects of the inventive concepts may be betterunderstood in connection with the following non-limiting examples.

EXAMPLES Example 1 In Vivo Study 1

A comparison was made of the safety, pharmacokinetics, aerosol deliverycharacteristics, and nebulization time of the conventional dose andinhalation delivery system (5 mL ampoule containing 300 mg tobramycinand 11.25 mg sodium chloride in sterile water for injection (TOBI®tobramycin solution for inhalation, Chiron Corporation, Seattle, Wash.),pH 6.0; administered with a PARI LC PLUS™ jet nebulizer with a PulmoAidecompressor) with 3 doses of TOBI (30 mg tobramycin in 0.5 mL solution,60 mg in 1.0 mL, and 90 mg in 1.5 mL) using a AeroDose™ inhaler device.

The study was designed as an open label, randomized, multicenter, singledose, unbalanced, four treatment, three period crossover trial. Eachpatient was to receive three single doses of aerosolized antibiotic: theactive drug control treatment during one treatment period and two ofthree experimental treatments during two additional treatment periods.Single dose administration during the three treatment periods was tooccur at one-week intervals.

In accordance with the study design, forty eight eligible male andfemale patients 12 years of age or older with a confirmed diagnosis ofcystic fibrosis were to be enrolled in the study and randomly assignedto one of 12 treatment sequences of three treatments each (one activecontrol and two experimental treatments) with the constraint that theactive control treatment was to be administered in either the first orthe second of the three treatment periods. Experimental treatments wereadministered during all three treatment periods. Each patient inhaled asingle dose of aerosolized control and two of three experimentaltreatments in accordance with the present invention as follows.

-   -   control delivery treatment (PARI LC PLUS jet nebulizer+PulmoAide        compressor):        -   TOBI 300 mg in 5 mL solution.    -   experimental delivery treatments (AeroDose™ inhaler breath        actuated nebulizer):        -   TOBI 30 mg in 0.5 mL solution;        -   TOBI 60 mg in 1.0 mL solution;        -   TOBI 90 mg in 1.5 mL solution.

The duration of study participation for each patient was to beapproximately five weeks including a brief (2 days to one week)screening period, three one-week treatment periods, and a one-weektelephone follow-up period.

Control and Experimental Treatments

Each patient was to self-administer under research staff supervision atotal of three single doses of aerosolized tobramycin during the study,one dose per crossover treatment period. Patients were to receive asingle dose of the control delivery treatment during period 1 or period2 of the three treatment periods. In addition, each patient was toreceive single doses of two of the three experimental deliverytreatments during the remaining two treatment periods. Control andexperimental delivery treatments were specified as follows.

Control Delivery Treatment:

PARI LC PLUS jet nebulizer with PulmoAide compressor: preservative freetobramycin 60 mg/mL (excipient 5 mL of ¼ normal saline adjusted to a pHof 6.0±0.5); 300 mg in 5 mL.

Experimental Delivery Treatments:

-   -   Aerodose with a 3-4 μm mass medium diameter (MMD) aerosol        particle size: preservative free tobramycin 60 mg/mL (excipient        0.5 mL of ¼ normal saline adjusted to a pH of 6.0±0.5); 30 mg in        0.5 mL;    -   Aerodose with a 3-4 μm MMD: preservative free tobramycin 60        mg/mL (excipient 1.0 mL of ¼ normal saline adjusted to a pH of        6.0±0.5); 60 mg in 1.0 mL;    -   Aerodose with a 3-4 μm MMD: preservative free tobramycin 60        mg/mL (excipient 1.5 mL of ¼ normal saline adjusted to a pH of        6.0±0.5); 90 mg in 1.5 mL.

Patients were placed upright in a sitting or standing position topromote normal breathing and were instructed to place the nose clipsover the nostrils and to breath normally through the mouth until therewas no longer any mist produced by the nebulizer. Aerosol delivery wasanticipated to take 15 minutes to complete.

A pharmacist or coordinator prepared the 30 mg dose of TOBI by drawing0.5 mL of the 60 mg/mL TOBI formulation into a one-mL syringe. Eachsyringe was labeled with the patient identification number. Study drugwas dispensed into the medication reservoir as indicated in the Aerodosedirections for use. TOBI 60 mg and 90 mg doses were similarly preparedby drawing two and three 0.5 mL aliquots, respectively, from the TOBIampoule into two and three one-mL syringes.

Aerosol Delivery Systems

The control delivery system (PARI LC PLUS jet nebulizer) was used onceper patient during the study for administration of TOBI 300 mg (controltreatment). The experimental delivery system (Aerodose inhaler) was usedto deliver only one dose of study treatments.

The control nebulizer, the PARI LC PLUS jet nebulizer with DeVilbissPulmoAide compressor, generates aerosol by air-jet shear. A detailedcomparison of experimental and control devices is provided in Table 1.

TABLE 1 DEVICE COMPARISON PARI LC PLUS Nebulizer and DeVilbiss PulmoAideDevice Characteristic Aerodose Nebulizer Compressor Aerosol generatingprinciple Piezoelectric vibration Air-jet shear Aerosol characteristicswith TOBI Mass median diameter (MMD) 4.0 μm 4.8 μm Output rate 8.0μL/sec 3.6 μL/sec Emitted dose 85% 57% Dose actuation Breath-actuated byuser On/off switch; when on, inhalation medication aerosolizedcontinuously Control of aerosol generation Breath actuated. AnContinuous aerosol output during airflow sensor system is bothinhalation and exhalation used to limit aerosol generation to inhalationUser indicator lights Green LED flashing for None “device ready” andsolid for “aerosolization” Red LED for “low battery” Physicalcharacteristics Size 3.3″ × 2.6″ × 1.1″ 7.5″ × 7.5″ × 3.0″ (nebulizer)10.1″ × 10.5″ × 6.5″ (compressor) Weight 140 gm 68 gm (nebulizer) 3,200gm (compressor) Power source Four AAA alkaline 115 VAC, 60 Hz batteriesPower consumption 2.5 watts 90 watts (max.) Where used Fully portableRestricted to power outlets supplying 115 VAC, 60 HzSelection of Doses in the Study

Commercial TOBI 60 mg/mL in 5 mL solution administered by PARI LC PLUSjet nebulizer and powered by the PulmoAide compressor was the activedrug control delivery system against which potential improvements inaerosol delivery technology by the Aerodose breath actuated nebulizerwere compared in this example.

The selection of doses of experimental treatments (TOBI 30 mg in 0.5 mLsolution, 60 mg in 1.0 mL, and 90 mg in 1.5 mL) was based on empiricaldata on the comparative predicted efficiency of the Aerodose inhalerrelative to the PARI LC PLUS nebulizer. The selection of doses was alsobased on the assumption that TOBI delivered via the PARI LC PLUS jetnebulizer leads to the systemic absorption of approximately 11.7% of theadministered dose {Pitlick, Nardella, et al., 1999}. Furthermore, themean and standard deviation of the serum concentration one hour afterinhalation was 1.0 μg/mL±0.58, suggesting a wide range of deposition(Table 5.2 C, Clinical Pharmacology, PathoGenesis NDA, #50,753). Due todesign features of the Aerodose inhaler, it was estimated that between50-70% of the drug would be delivered to the lung. This assumption isbased on the predicted efficiency of a nebulized dose.

Patients were randomized to treatment sequence groups, and predoseprocedures were completed including a physical examination (only ifabnormal during screening), recheck of inclusion and exclusion criteria,interim history review, spirometry, clinical evaluation, and blood andurine specimens for laboratory tests (only if abnormal duringscreening). A bronchodilator was to be administered before dosing ifregularly used by the patient. Spirometry was completed 15-60 minutesafter the bronchodilator, if applicable.

Patients received a single dose of study treatments during each of threetreatment periods separated by an interval of 7 days between treatments.At the time of single dose administration during each period, patientswere instructed to sit upright and use nose clips during aerosol doseadministration.

Patients remained at the clinic through completion of 8-hour posttreatment procedures (nebulization time, spirometry, and sputum, serumand urine specimens for tobramycin determinations). Patients were thendischarged from the clinic and were expected to collect and return their8-24 hour urine collection at the next visit, no later than 7 days aftertheir previous visit. Patients were to refrigerate urine collections atall times except during transport.

Safety Variables

Safety was assessed by monitoring the incidence of bronchospasm and bythe quantitative change in pulmonary function (measured as change inFEV₁% predicted), the incidence of treatment emergent adverse events,and the incidence of unusually high serum tobramycin results (≧4 μg/mL),the significance of clinical laboratory test results, and thesignificance of change in clinical evaluation results.

Bronchospasm (Airway Reactivity)

One objective of the study was to compare the rate of occurrence ofbronchospasm (airway reactivity) between control and experimentaldelivery systems. Bronchospasm was measured by the change in forcedexpiratory volume in 1 second [FEV₁ (liters)] from before dosing to 30minutes after dosing during periods 1, 2, and 3. The number and percentof patients who experienced predose to postdose decreases in FEV₁(liters) that were ≧10% and those that were ≧20% were determined toassess the comparative incidence of bronchospasm among control andexperimental treatments. Decreases in FEV₁ (liters) that were ≧20% wereconsidered clinically significant for the purposes of the study.Additionally, an acute decrease in FEV₁ (liters)≧30% from before toafter treatment was considered a symptom of respiratory distress. Inthis event, continuation of the patient in the study was at thediscretion of the investigator.

Norms have been developed for FEV₁. These norms are commonly used instudies of pulmonary patients. This study employed the Knudson equationsthat use age, gender, and height to predict a patient's FEV₁ values asif the patient was free of pulmonary function disease. The actual FEV₁value is divided by the normative value, and the resulting ratio ismultiplied by 100 to produce a measure that represents percentage ofpredicted normal function, commonly called percent predicted. Thetransformation is:FEV₁% predicted=(FEV_(1 actual value)/FEV_(1 normative value))×100

Relative change in FEV₁% predicted is defined as the percent change frompredose to 30 minutes postdose in FEV₁% predicted and is calculated as:

relative change inFEV₁%predicted=[(FEV_(1 (% predicted at 30 minutes postdose))−FEV_(1 (% predicted at postdose)))/FEV_(1 (% predicted at predose))]×100Clinical Laboratory Tests

Serum creatinine, blood urea nitrogen (BUN), and dipstick urine proteinresults were obtained from specimens drawn during screening and beforedosing during treatment period 3. Urine dipstick testing was alwaysperformed on fresh specimens. Serum and urine specimens that needed tobe retained at the site (e.g., those drawn after shipping pick-up hoursor on Friday or Saturday) were frozen until shipment at the nextearliest shipping time. Specimens were covered with dry ice forshipping.

All out of range laboratory results were evaluated for clinicalsignificance and drug relationship by the investigator using thefollowing classification scheme:

-   -   clinically insignificant;    -   possible study medication relationship;    -   probable study medication relationship;    -   unrelated to study medication, related to concurrent illness;    -   unrelated to study medication, related to other concurrent        medication;    -   other (investigator commentary).        Aerosol Delivery Variables

Evaluation of the aerosol delivery characteristics of the Aerodosebreath actuated nebulizer, compared to characteristics of theFDA-approved PARI LC PLUS jet nebulizer with PulmoAide compressor, wasbased on determination of sputum, urine, and serum tobramycinconcentrations, calculation of certain sputum and serum pharmacokineticparameters, and measurement of nebulization time.

Sputum Tobramycin Concentrations

Before study treatments were administered, patients expectorated sputumproduced from a deep cough into an individual specimen container.Immediately after treatment, patients rinsed their mouths three timeswith 30 mL of normal saline, gargled for 5-10 seconds, and expectoratedthe rinse.

Post treatment sputum specimens were collected following the normalsaline gargle at 10 minutes and at 1, 2, 4, and 8 hours after completionof the aerosol drug administration for determination of sputumtobramycin concentrations. Sputum specimens were judged to be acceptableif collected within ±2 minutes of the scheduled 10-minute posttreatmentcollection time and within ±10 minutes of the scheduled 1-, 2-, 4-, and8-hour collection times. After collection, specimens were immediatelyfrozen for later determination of tobramycin concentrations in sputum. Aminimum of 1 gram of sputum was required for analysis. Tobramycinconcentrations in sputum (sputum LOQ=20.0 μg/gm) were measured by usingHPLC.

Serum Tobramycin Concentrations

Whole blood was drawn by venipuncture, an indwelling heparin/salinelock, or a permanent venous access port at 10 minutes and at 1, 2, 4,and 8 hours after completion of dosing. Blood specimens were judged tobe acceptable if collected within ±2 minutes of the scheduled 10-minuteposttreatment collection time and within ±10 minutes of the scheduled1-, 2-, 4-, and 8-hour collection times. Blood specimens were allowed toclot for 30 minutes and were then centrifuged at 1500×g for 10 minutesuntil clot and serum separated. Serum samples (3 mL) were pipetted intoplastic vials and frozen immediately for later determination of serumtobramycin concentrations.

Tobramycin concentrations in serum were measured by Abbott TDxFLx® assay(Abbott Laboratories, Abbott Park, Ill.) [serum lower limit ofquantitation (LOQ)=0.18 μg/mL].

Urine Tobramycin Recovery

Urine specimens were collected and combined in a 24-hour collectioncontainer during the 12 hours before treatment (-12-0 hour period) andduring 0-8 hour and 8-24 hour collection periods after treatmentaccording to instructions provided in the Study Manual. Total urinevolume for the collection period was recorded, and a 10 mL aliquot fromeach urine collection was retained and frozen for later analysis ofurine tobramycin concentration.

The recovery of tobramycin in urine (in milligrams) during 0-8 hour and8-24 hour collection periods was calculated as follows.

 urine tobramycin recovery (μg)=urine volume (mL)·urine tobramycinconcentration (μg/mL)

Urine tobramycin recovery was normalized for each collection periodaccording to TOBI dose as follows.dose-normalized urine tobramycin recovery (μg/mg)=[urine tobramycinrecovery (μg)÷TOBI dose (mg)]

The percent of the TOBI dose excreted in urine in the 24-hour periodfollowing treatment was calculated as follows.% tobramycin excreted in urine=[(urinary recovery in μg÷1000 μg/mg)+TOBIdose in mg]·100%.

If either the urine volume or the urine tobramycin concentration for acollection interval was missing, then the urine tobramycin recovery wasnot calculable for that interval. If calculated urine tobramycinrecovery was missing for either the 0-8 hour or the 8-24 hour collectioninterval, then the 0-24 hour urine tobramycin recovery was notcalculated. Missing urine tobramycin recovery values were not replacedby estimated values for analysis purposes.

Tobramycin concentrations in urine were measured by Abbott TDxFLx® assay[urine lower limit of quantitation (LOQ)=1.0 μg/mL].

Pharmacokinetic Parameters

The maximum tobramycin concentrations (C_(max)) in sputum and serumduring the 8-hour posttreatment sampling period were identified for eachpatient during each treatment period, and the time at which C_(max) wasobserved (T_(max)) was recorded.

Area under the concentration-time curve through 8 hours postdose(AUC₀₋₈) was calculated from sputum and serum tobramycin concentrationsusing the linear trapezoidal method. Nebulization time (excluding timefor refilling) was added to the time between predose and 10 minutespostdose for AUC₀₋₈ calculations.

Area under the concentration-time curve extrapolated to infinity(AUC_(0-∞)) was calculated for sputum and serum as follows.AUC_(0-∞)=AUC_(0-last) +C _((last)) ÷k _(el)

-   -   where: AUC_(0-last) is area under the curve from predose through        the last non-BQL time        -   C_((last)) is the last non-BQL posttreatment concentration            result        -   k_(el) is the elimination rate constant (terminal phase            slope) and k_(el)=log 2÷T_(1/2)    -   where T_(1/2) is the elimination half-life for the patient.

Relative systemic bioavailability was calculated based on serum AUC₀₋₈values for control (TOBI 300 mg delivered by PARI LC PLUS nebulizer) andexperimental (TOBI 30 mg, 60 mg, and 90 mg delivered by Aerodoseinhaler) groups as follows.relative bioavailability (%)=experimental group serum AUC₀₋₈÷controlgroup serum AUC₀₋₈

Missing tobramycin concentrations and those reported as zero or belowquantifiable limits (BQL) were not to be replaced with any estimatedvalue. C_(max) and AUC₀₋₈ were always determinable except in the eventthat all posttreatment tobramycin concentrations were missing, zero, orBQL. There was no missing sputum C_(max) and AUC₀₋₈ values among the 49patients who completed the study (refer to report section 9.3.1 fordetails). Four completing patients had indeterminate serum C_(max) andAUC₀₋₈ values due to BQL serum results for each posttreatment samplingtime (refer to report section 9.4.1 for details).

Nebulization Time

The timing (duration) of nebulization began with the patient's firsttidal breath after the device was in place and continued until thedevice aerosolized no more TOBI solution. Nebulization time did notinclude any interruptions or time needed for instillation of drug intothe nebulizer between the repeat filling of the AeroDose™ inhaler. Thelength of any interruption in nebulization and the reason forinterruption were recorded.

Safety Analyses

Reductions in FEV₁% predicted ≧10% and ≧20% were used as indicators ofthe occurrence of bronchospasm (airway reactivity). McNemar's test forpaired comparisons (replacing the Cochran-Mantel-Haenszel (CMH) test)was used for control vs. experimental treatment comparisons of theincidence of patients with predose to 30-minute postdose decreases inFEV₁% predicted that were ≧10% and ≧20%. In addition, pairwise t-testswere used to compare mean relative change in spirometry FEV₁% predictedfrom predose to postdose between each experimental treatment and thecontrol treatment. All statistical analyses were performed usingtwo-sided tests conducted at a 0.05 significance level (i.e., α=0.05).Since all statistical tests were exploratory in nature, no adjustment ofp-values was made for multiple testing. Changes from predose to postdosein vital signs, body weight, and the incidence of abnormal and/orclinically significant laboratory and physical examination results weresummarized and evaluated descriptively.

Individual patient serum tobramycin results were monitored for unusuallyhigh values (≧4 μg/mL) that might potentially indicate the occurrence ofsystemic toxicity.

Aerosol Delivery Analyses

The natural logarithms of AUC₀₋₈, AUC_(0-∞), and C_(max) based on sputumand serum tobramycin concentrations were to be statistically analyzedusing a mixed-effect repeated-measure analysis of variance modelcontaining treatment, sequence, period, and carryover as fixed effectsand patient as a random effect. In the planned analysis of variancemodel, sequence and carryover (treatment by period interaction) effectswere confounded. The actual model used for the analysis was thereforemodified by dropping the sequence term so that the assessment ofcarryover (i.e., treatment by period interaction) could proceed. WhenAUC_(0-∞) values were calculated, large outlier values were noted, andthe analysis for this parameter was dropped.

Three hypotheses regarding whether the experimental delivery treatmentof 30 mg, 60 mg, or 90 mg TOBI was equivalent to the control deliverytreatment of 300 mg TOBI were to be tested in the model. Theexperimental treatment to control ratio for each of the log AUC andC_(max) parameters was estimated with 90 percent confidence intervals(CIs). Upper and lower limits for the CIs were then obtained by backtransformation (i.e., by exponentiating the log values of the upper andlower limits) to the original scale of the parameter. If the CIs for theratio of experimental and control treatments contained the value of 1.0,it was concluded that the treatments were not significantly different atthe α=0.1 for the 90% CIs.

If demographic or baseline characteristics showed important apparentdifferences between the three experimental AeroDose™ groups compared toall patients, then the discrepant factor and its interaction with thedelivery treatment factor were to be added to the mixed-effect model.Exploratory evaluations of age, gender, body weight, and baselinepulmonary function (FEV₁ percent predicted) demonstrated no importanteffects on pharmacokinetic results.

Disposition of Patients

A total of 56 patients were screened for the study by the nineinvestigators. Fifty-three patients met entrance criteria, were enrolledin the study, and were randomized to one of the 12 sequences oftreatment administration identified in the randomization code. A totalof 3 patients failed to meet entrance criteria and were not enrolled inthe study: 2 patients had screening FEV₁% predicted results that werebelow the 40% criterion required for entry, and one patient exhibiteddisqualifying serum creatinine, BUN, and/or proteinuria.

Accrual of the 53 randomized patients at 9 sites was as follows: 3 sitesrandomized 8 patients each, 2 sites randomized 7 patients, 3 sitesrandomized 4 patients, and one site randomized 3 patients. Fifty twopatients received at least one dose of study treatments, and one patientwas enrolled and randomized but withdrew from the study before the firststudy treatment due to increased productive cough with a significantdecline in forced expiratory volume (FEV) since screening (both eventsand associated hyperventilation were considered SAEs due tohospitalization of the patient: included in study database).

Of the 52 patients who received study treatments, 49 patients completedthe study, and 3 patients withdrew after having received one dose ofstudy treatment. Two of the withdrawn patients discontinued the studyduring the control treatment period (TOBI 300 mg administered by PARI LCPLUS nebulizer), and one patient withdrew during the TOBI 90 mg byAeroDose™ inhaler treatment period.

Baseline Characteristics

Enrolled patients had documented laboratory (sweat chloride≧60 mEq/L byquantitative pilocarpine iontophoresis test (QPIT) and/or genotype with2 identifiable mutations) and clinical evidence consistent with adiagnosis of cystic fibrosis. Patients met all inclusion and exclusioncriteria except for one patient whose pulmonary function entrancerequirement (FEV₁≧40% of predicted based on gender, age, and height) waswaived (the patient's screening FEV₁% predicted was 39.87%). The averageFEV₁% predicted of all randomized patients was 66.4% at screening with arange from approximately 40% to 116%.

Patients reported no known local or systemic hypersensitivity toaminoglycosides. Patients had taken no loop diuretics, no form ofaminoglycoside within 7 days before study treatments, and noinvestigational medications within 2 weeks before study treatments.

Female patients had a negative pregnancy test before study treatments,and all patients had serum creatinine≦2.0 mg/dL, BUN<40 mg/dL, and <2+proteinuria at visit 1 screening, as required by the protocol. Screeningor repeat serum creatinine and BUN results were within the normal rangesfor these tests before study treatments. Screening or repeat urineprotein results were positive 1+ in 3 patients, but this result did notpreclude participation of these patients in the study.

No disqualifying medical history or physical examination findings werenoted at visit 1 screening. Screening and visit 1 predose vital signswere unremarkable for nearly all patients. One patient exhibited lowsystolic and diastolic blood pressures at (72/49 mmHg), but theseresults did not preclude participation of the patient in the study.

Safety Evaluation

Extent of Exposure

Forty-nine patients received all 3 single doses of study treatmentsaccording to the randomization code, and 3 patients who withdrew fromthe study received one dose of study treatment. These 52 patients wereincluded in the safety evaluation. Fifty-one of the 52 patients receiveda single dose of TOBI 300 mg, and 34, 32, and 33 of the 52 patientsreceived a single dose of TOBI 30 mg, 60 mg, and 90 mg, respectively.Three of the 49 completing patients had to stop treatment due to inhalermalfunction and subsequently repeated the treatment period at a laterdate. As a result, these 3 patients received a partial dose of TOBIduring the period in which the malfunction occurred (the amount of thepartial dose was not recorded) and a full dose of TOBI during therepeated period.

Pulmonary Function Results

Bronchospasm

In one aspect, the study compared the rate of occurrence of bronchospasm(airway reactivity) between control and experimental delivery systems.The occurrence of bronchospasm was determined quantitatively based onthe percent change in FEV₁ (liters) from before dosing to 30 minutesafter dosing in each of the 3 treatment periods. For the purposes of thestudy, predose to postdose reductions in FEV₁ (liters)≧10% and ≧20% weredefined as bronchospasm; reductions in FEV₁ (liters)≧20% were consideredclinically significant.

Fifteen patients (9 male and 6 female) experienced 24 instances ofbronchospasm during the study. Two instances of clinically significantbronchospasm were observed (decline in FEV₁ (liters)≧20%: patient105-1034 after TOBI 300 mg and patient 102-1040 after TOBI 60 mg). Nostatistically significant pairwise differences in the overall incidenceof bronchospasm were noted between control and experimental treatments.No clear relationship appeared to exist between the incidence ofbronchospasm and TOBI dose or delivery system (see Table 2 below).

TABLE 2 Incidence of Acute Bronchospasm by Treatment TOBI TOBI TOBI 30mg 60 mg 90 mg TOBI 300 mg Aerodose Aerodose Aerodose Bronchospasm PARILC PLUS¹ inhaler² inhaler² inhaler² Parameter (N = 51) (N = 34) (N = 32)(N = 33) FEV₁ 9 (17.6%) 5 (14.7%) 6 (18.8%) 4 (12.1%) Decrease ≧ 10%FEV₁ 1 (2.0%) 0 (0.0%) 1 (3.1%) 0 (0.0%) Decrease ≧ 20% Bronchospasm wasdefined by protocol as a decrease in FEV₁ (liters) ≧ 10% and ≧ 20% frompredose to 30 minutes postdose. Declines ≧ 20% were consideredclinically significant. ¹Control (C) treatment = TOBI 300 mg deliveredby PARI LC PLUS nebulizer. ²Experimental (E) treatments = TOBI 30, 60,or 90 mg delivered by Aerodose inhaler.

One patient 34 experienced clinically significant bronchospasm at 30minutes after completing the TOBI 300 mg dose during treatment period 1(visit 2). This 32-year old male patient's FEV₁ was 2.55 L before dosingand 1.98 L (decline in FEV₁ (liters)≧20%) at 30 minutes after dosing. Heexperienced moderate chest tightness that resolved spontaneously. Thispatient also experienced a second episode of bronchospasm 30 minutesafter TOBI 60 mg during period 2. The FEV₁ was 2.47 L before dosing and2.14 L (decline in FEV₁ (liters)≧10% but <20%) at 30 minutes afterdosing. No symptomatology was reported at the time of this event. Noprestudy aminoglycoside use was noted for this patient.

One patient experienced one instance of clinically significantbronchospasm 30 minutes after TOBI 60 mg during period 3 (visit 4) ofthe crossover. This 36-year old male patient's FEV₁ was 2.26 L beforedosing and 1.75 L (decline in FEV₁ (liters)≧20%) at 30 minutes afterdosing (Archival Listing 3), but he reported no other symptomatology atthis time. No prestudy aminoglycoside use was noted for this patientThis episode of bronchospasm appeared due in part to anuncharacteristically high predose FEV₁ value. The 30-minuteposttreatment value was similar to that obtained during period 2 whenthe change in FEV₁ did not meet the definition of bronchospasm.

Among the 13 patients who experienced clinically non-significantbronchospasm, one patient experienced a decline in FEV₁ (liters)≧10% but<20% after all three study doses were administered, 6 patientsexperienced a decline in FEV₁ (liters)≧10% after two doses of studymedication, and 6 patients experienced a single instance ofbronchospasm. Table 3 below lists instances of bronchospasm by patient,treatment period, and TOBI dose.

TABLE 3 Patient Dosing Regimen and Acute Bronchospasm Site- Period 1(Visit 2) Period 2 (Visit 3) Period 3 (Visit 4) Patient ID/Gender TOBIDose Received TOBI Dose Received TOBI Dose Received 108-1048^(b)/Female300^(c)  30^(c)  60 109-1015^(b)/Male 300  30^(c)  60 107-1027/Male 300 30^(c)  90^(c) 103-1038^(b)/Female 300^(c)  60  30 105-1034/Male300^(d,e)  60^(c)  30 107-1026/Female 300  60^(c)  90102-1009^(b)/Female 300^(c)  90^(c)  30 102-1040^(b)/Male 300  90 60^(d) 106-1050^(b)/Female  30^(c,e) 300  90 102-1007^(b)/Male  60^(c)300^(c)  30 104-1021/Male  60^(c) 300^(c)  30 108-1044/Male  60 300^(c) 30^(c) 105-1047/Female  60 300^(c)  90 106-1022^(b)/Male  90^(c,e) 300 30 106-1041^(b)/Male  90^(c,e) 300^(c)  60^(c) Bronchospasm is definedas a decrease in FEV₁ (liters) ≧ 10% and ≧ 20% from predose to 30minutes postdose. Declines ≧ 20% were considered clinically significant.^(b)The patient used a bronchodilator before dosing with studymedication. ^(c)Bronchospasm (not clinically significant): decrease inFEV₁ (liters) ≧ 10% but < 20%. ^(d)Bronchospasm (clinicallysignificant): decrease in FEV₁ (liters) ≧ 20%. ^(e)The patient alsoreported “lung function decrease” (COSTART term) as an AE during thedesignated treatment period.

Three of the 15 patients with bronchospasm reported treatment-relatedsymptoms at the same time. One patient 15 experienced moderate wheezing(coded as asthma) after TOBI 30 mg during period 2, one patient 4experienced moderate chest tightness (coded as chest pain as reportedpreviously) after TOBI 300 mg during period 1, and one patient 41experienced increased cough after TOBI 60 mg during period 3. All eventsresolved either spontaneously (chest tightness), with treatment(wheezing), or by holding and restarting therapy (increased cough). Noneof the adverse events led to a serious outcome.

Four of the 15 patients with bronchospasm (and one patient withoutbronchospasm) reported “lung function decreased” (COSTART term) as anadverse event. In addition to the 4 patients with bronchospasmidentified in Table 3 above, one patient who experienced nobronchospasm, reported lung function decreased once after TOBI 60 mg andonce after TOBI 90 mg delivered by the AeroDose™ inhaler.

Initial instances of bronchospasm occurred more frequently during period1 than during periods 2 or 3 of the crossover. Nine of the 15 patientsfirst experienced bronchospasm during the first treatment period (visit2), five patients during the second treatment period, and one patientduring the third treatment period.

Patients who routinely used a bronchodilator were permitted to continueto do so during the study. Bronchodilator doses were to be administered15 to 60 minutes prior to study treatments. Nine of the 15 patients whoexperienced bronchospasm during the study used a bronchodilator prior toadministration of study treatment.

Relative Change in FEV₁% Predicted

The magnitude of the relative change in FEV₁% predicted was calculatedas a quantitative measure of the effect of TOBI treatments on pulmonaryfunction during the study. There were no statistically significantdifferences among the 4 treatments and no evidence of the presence ofperiod or carryover (treatment by period interaction) effects. Resultsof pairwise comparisons between control and experimental treatments aresummarized in Table 4. Since the overall treatment difference was notstatistically significant, the significant p-value for the TOBI 300 mgvs. TOBI 30 mg comparison in Table 4 below (p=0.019) should not beinterpreted as conclusive evidence of a difference. FIG. 1 graphicallyillustrates the mean relative changes in FEV₁% predicted from before to30 minutes after dosing for each of the treatments.

TABLE 4 MEAN (SD) RELATIVE CHANGE IN FEV₁ % PREDICTED FEV₁ % TOBI 300 mgTOBI 30 mg TOBI 60 mg TOBI 90 mg Predicted (%) PARI LC PLUS¹ Aerodoseinhaler² Aerodose inhaler² Aerodose inhaler² Parameter (n = 51) (n = 34)(n = 32) (n = 33) Predose 67.8 (18.4) 65.5 (17.1) 65.4 (16.8) 71.3(20.0) n = 51 n = 34 n = 32 n = 33 30 minutes 63.7 (17.6) 63.0 (16.7)62.5 (15.7) 68.7 (19.1) postdose n = 51 n = 34 n = 32 n = 32 Relativechange −6.1 (5.2) −3.8 (5.4) −4.2 (6.2) −3.2 (7.4) from predose³ n = 51n = 34 n = 32 n = 32 P-value for Treatment: 0.141 Period: 0.199Carryover: NC crossover: Pairwise contrasts: 0.019 0.058 0.083 C vs. Ep-value (paired t-test): ¹Control (C) treatment = TOBI 300 mg deliveredby PARI LC PLUS nebulizer. ²Experimental (E) treatments = TOBI 30, 60,or 90 mg delivered by Aerodose inhaler. ¹Relative change from predose =100% · ((30 minute postdose value − predose value)/predose value). NC =carryover (treatment by period interaction) effect not statisticallysignificant and was dropped from final model.Safety Conclusions

Nine males and six females experienced treatment-induced bronchospasmduring the study. There was no difference in the rate of occurrence ofTOBI induced bronchospasm between control and experimental deliverysystems regardless of dose. The occurrence of bronchospasm was rarelyassociated with patient symptoms. All but four of the patientsexperiencing drug-induced bronchospasm had been prescribedbronchodilators prior to the study suggesting that they had a history ofairway reactivity. The disproportionate number of males versus femalesexperiencing airway reactivity is unusual in light of the fact thatenrollment was approximately 60% female and 40% male. The pivotal trialsshowed that gender had no influence on drug induced airway reactivity.However, it would be difficult to base any conclusions on this findingdue to, the small patient numbers in this study.

Treatment-emergent adverse events occurred in all treatment groupsregardless of causality. The most common treatment-emergent experienceswere associated with Respiratory and Body as a Whole systems. The mostcommon individual events were cough increased, rhinitis, sputumincreased, asthma, chest pain, and headache. These events were alsocommon to the patient's pretreatment symptoms reflecting the patientsunderlying disease. For the majority of treatment-emergent adverseevents, there were no meaningful differences between TOBI doses orbetween the PARI LC PLUS nebulizer and the AeroDose™ inhaler.

The serious adverse events (SAEs) reported were primarily associatedwith an exacerbation of the patients underlying disease states. The onetreatment-related SAE involved a possible sensitivity reaction that, ifdocumented, would have occurred regardless of device or dose.

Review of the clinical chemistry, vital signs, and physical findings didnot reveal any clinically significant safety issues associated with thedose or delivery system used to administer TOBI.

All the patients were on multiple concurrent medications appropriate totheir disease state (cystic fibrosis), other underlying illnesses, andage throughout the study. The concurrent medications did not appear tohave any influence on the safety profile of the study drug or eitherdevice during the study. Overall, no clinically significant orunexpected safety issues for TOBI were identified in the study. Thestudy showed that there were no meaningful differences in the safetyprofiles of administering TOBI via the PARI LC PLUS delivery system incomparison with the Aerodose delivery system regardless of dose.

Aerosol Delivery Results

Data Analysis

Forty-nine of the 52 dosed patients completed the study and wereevaluable for pharmacokinetics by reason of having completed at least 2doses of study treatments. These 49 patients also constituted the“completers” subset of patients referred to in the summary tables. Threeof the 52 dosed patients discontinued the study before completing 2doses of study treatments and were not evaluable for pharmacokinetics.All 52 patients were evaluable for the aerosol delivery objective(nebulization time) of the study.

Sputum Tobramycin Concentrations and Pharmacokinetic Parameters

Compliance with Specimen Collection Requirements

Six of 49 completing patients had a total of 11 missing sputumspecimens. No more than one sputum sample was missed per treatment-time(e.g., for TOBI 300 mg at one hour postdose). Two patients missed 2 ormore sputum samples during the study, and four patients missed a singlesputum sample.

A single completing patient provided no sputum pharmacokinetic data forthe TOBI 60 mg treatment. One patient had missing sputum samples from 10minutes through 8 hours after TOBI 60 mg treatment. After the databasewas locked, the missing sputum concentration results were located.Sputum tobramycin concentrations at 10 minutes and 1, 2, 4, and 8 hourswere 0.82 μg/gm, BQL, 0.0, 0.0, and 0.0, respectively. The database wasnot subsequently unlocked to add these data, since the inclusion ofthese values would have had minimal impact on estimation and analyses ofpharmacokinetic parameters. As a result, only C_(max) (0.82 μg/gm) andT_(max) (10 minutes=0.17 hour) values were excluded from TOBI 60 mg PKestimates and analyses; AUC values were incalculable due to BQLtobramycin concentrations from one through 8 hours after TOBI 60 mgtreatment.

Sputum Tobramycin Concentrations

Pretreatment sputum tobramycin concentrations for all completingpatients were below the limit of quantifiability (LOQ) throughout thestudy.

After dosing, sputum concentrations increased rapidly, reaching maximumconcentrations within 10 minutes (see FIG. 2), and declined thereafterwith median half-life values ranging from approximately 1.6 to 2.1 hoursduring the four treatments. The sputum concentrations were highlyvariable among patients, as coefficients of variation (standarddeviation divided by the mean times 100%) approached or exceeded 100%for each treatment at all time points.

For the AeroDose™ inhaler, mean sputum tobramycin concentrationsincreased with increasing TOBI dose at each measurement time during the8-hour postdose period. Mean sputum concentrations for the TOBI 90 mgtreatment with the AeroDose™ inhaler were similar throughout the 8-hourperiod to those obtained for the TOBI 300 mg treatment with the PARI LCPLUS nebulizer.

By 2 hours after the end of TOBI 30 mg and by 8 hours after TOBI 60 mg,90 mg, and 300 mg treatments, sputum concentrations were below LOQ in atleast half of the patients. Period effects on sputum tobramycinconcentrations were not observed.

After TOBI administration using the AeroDose™ inhaler, maximum plasmaconcentrations (C_(max)) and area under the plasma concentration timeprofile (AUC₀₋₈) increased linearly with dose (Table 5 below and FIGS. 3and 4), suggesting linear pharmacokinetics. Dose normalized C_(max) andAUC values were comparable among AeroDose™ dose levels, indicating doseproportionality (based on AUC values).

Comparing devices, mean C_(max) and AUC₀₋₈ for the TOBI 90 mg treatmentdelivered by the AeroDose™ inhaler achieved similar levels as thoseobtained by the TOBI 300 mg treatment delivered by the PARI LC PLUSnebulizer. The dose normalized C_(max) and AUC₀₋₈ results were higherduring AeroDose™ treatments than during the PARI LC PLUS treatment,indicating that the AeroDose™ inhaler exhibited higher efficiency. Thebioavailability of the AeroDose™ device was about 3-fold higher thanthat of the PARI LC PLUS nebulizer.

Exploratory analyses suggested that sputum pharmacokinetic results wereunaffected by characteristics present before treatments began (age,gender, body weight, FEV₁% predicted at screening) and were unaffectedby events noted after the start of treatments (device failure,occurrence of bronchospasm defined as a decrease ≧10% in FEV₁, andrelative change in FEV₁% predicted).

TABLE 5 MEAN (SD) SPUTUM TOBRAMYCIN PHARMACOKINETIC PARAMETERS TOBI 30mg TOBI 60 mg TOBI 90 mg Sputum TOBI 300 mg Aerodose Aerodose AerodosePharmacokinetic PARI LC PLUS^(a) inhaler^(b) inhaler^(b) inhaler^(b)Parameters (n = 49) (n = 34) (n = 32) (n = 32) C_(max) (μg/gm) 985.65(839.34) 329.05 (311.30) 577.83 (538.42) 958.00 (952.30) No. pts withdata: 49 34 31 32 E vs C p-value^(c): <0.001 0.002 0.856 E/C (90%CIs)^(d): (0.23, 0.41) (0.43, 0.75) (0.72, 1.30) Dose-normalized Cmax(μg/gm)/mg 3.29 (2.80) 10.97 (10.38) 9.63 (8.97) 10.64 (10.58) No. ptswith data: 49 34 31 32 E/C (90% CIs)^(d): (2.82, 5.13) T_(max) (hr) 0.26(0.38) 0.24 (0.24) 0.38 (0.76) 0.33 (0.41) No. pts with data: 49 34 3132 T_(1/2) (hr) 6.41 (24.09) 2.04 (1.31) 12.89 (42.61) 13.02 (36.91)Median T_(1/2) (hr) 1.71 1.78 2.06 1.60 No. pts with data: 41 15 21 24AUC₀₋₈ (hr · μg/gm) 1471.16 (1278.22) 360.79 (422.23) 804.78 (722.83)1275.23 (1358.52) No. pts with data: 49 34 31 32 E vs C p-value^(c):<0.001 <0.001 0.465 E/C (90% CIs)^(d): (0.19, 0.28) (0.45, 0.69) (0.72,1.14) Dose-normalized AUC₀₋₈ 1.90 (4.26) 12.03 (14.07) 13.41 (12.05)14.17 (15.10) (hr · μg/gm)/mg No. pts with data: 49 34 31 32 E/C (90%CIs)^(d): (2.78, 4.12) AUC_(0-∞) (hr · μg/gm) 1996.36 (2013.70) 638.68(586.85) 1661.66 (2334.89) 5544.88 (14831.0) No. pts with data: 41 15 2124 ^(a)Control (C) treatment = TOBI 300 mg delivered by PARI LC PLUSnebulizer. ^(b)Experimental (E) treatments = TOBI 30, 60, or 90 mgdelivered by Aerodose inhaler. ^(c)Pairwise contrast: TOBI 300 mg PARILC PLUS group vs TOBI (30, 60, 90 mg) Aerodose groups.^(d)Back-transformed 90% confidence intervals around the mean of the logratio of E and C treatments. Sputum limit of quantifiability (LOQ): 20μg/gm.

Differences among the treatment groups in C_(max) and AUC₀₋₈ (Table 5above; FIGS. 3 and 4) were statistically significant (p<0.001) with noevidence of period or carryover (treatment by period interaction)effects. In pairwise comparisons, C_(max) and AUC₀₋₈ were significantlygreater for TOBI 300 mg than for TOBI 30 mg and for TOBI 60 mg but notfor TOBI 90 mg (90% CIs for C_(max)=(0.72, 1.30); for AUC₀₋₈=(0.72,1.14)).

The AeroDose™ inhaler was more efficient, regardless of TOBI dose, thanthe PARI LC PLUS nebulizer based on dose normalized sputum C_(max) andAUC₀₋₈ results. Dose normalized means for these pharmacokineticparameters were similar among AeroDose™ treatments but approximately3-fold higher than the dose normalized results after TOBI 300 mgdelivered by the PARI LC PLUS nebulizer (see Table 5).

The time to maximum sputum tobramycin concentrations (T_(max) in Table 5above) was similar for all treatment groups and averaged between 0.24and 0.38 hours for AeroDose™ doses compared to 0.26 hours for the TOBI300 mg treatment using the PARI LC PLUS. Elimination half-life (medianT_(1/2) in Table 5) was also similar among AeroDose™ treatments,averaging 1.60 to 2.06 hours, compared to 1.71 hours for TOBI 300 mg.

Exploratory analyses revealed no substantial association between sputumpharmacokinetic results and patient characteristics present beforetreatments (age, gender, body weight, pulmonary function [FEV₁%predicted] at screening) or emergent events after the start oftreatments (device failure, occurrence of bronchospasm [decrease≧10% inFEV₁ from predose to 30 minutes postdose], relative change in FEV₁.

Serum Tobramycin Concentrations and Pharmacokinetic Parameters

Forty-four (44) of 49 completing patients had no measurable serumtobramycin concentrations before dosing in any of the 3 treatmentperiods, and five patients exhibited measurable predose serum tobramycinabove the lower LOQ in the periods indicated in Table 6 below.

TABLE 6 MEASURABLE TOBRAMYCIN IN PREDOSE SERUM SPECIMENS Measurable^(b)Predose Previous Treatment Period Tobramycin during 8-hour Serum PeriodListed -- Tobramycin Serum Tobramycin Treatment^(a) TOBI DoseConcentration T_(1/2) Concentration Patient Sequence (mg) (μg/mL) (hr)(μg/mL) 107-1030 C-1-2 prestudy na^(c) na^(c) Per 1 -- 0.70 107-1027C-1-3 300 <0.20 1.68 Per 2 -- 0.29 105-1034 C-2-1 prestudy na^(c) na^(c)Per 1 -- 0.28 300 1.00 7.75 Per 2 -- 0.23 103-1019 1-C-2 30 0.35 10.85Per 2 -- 0.20 102-1007 2-C-1 prestudy na^(c) na^(c) Per 1 -- 0.77 600.75 7.71 Per 2 -- 1.38 300 0.96 10.62 Per 3 -- 0.60 ^(a)Treatments: C =Control TOBI 300 mg using PARI LC PLUS; 1 = TOBI 30 mg using Aerodoseinhaler; 2 = TOBI 60 mg using Aerodose inhaler; 3 = TOBI 90 mg usingAerodose inhaler. ^(b)Measurable tobramycin in serum: tobramycinconcentration > LOQ (0.2 μg/mL). ^(c)na = not available before the startof Period 1.

Table 6 also identifies predose serum specimens for periods 2, 3, orboth that bad measurable tobramycin in 4 of the 5 patients. Thesefindings are also reflected in non-zero mean amounts of predosetobramycin concentrations in periods 2 and 3. Three of the 5 patientsexhibited measurable serum tobramycin after having received TOBI 300 mgduring the immediately preceding study period.

These measurable predose results may represent carryover from previoustreatment or non-specific assay interference, but the low frequency andmagnitude of the results suggests that a substantial effect onposttreatment analyses was unlikely.

After each of the four TOBI treatments, serum tobramycin concentrationsgradually increased, reaching a maximum at one hour after dosing (FIG.5), and declined thereafter with median half-lives ranging from 2.73 to4.27 hours (Table 7 below).

For the Aerodose inhaler, mean serum tobramycin concentrations increasedwith increasing TOBI dose at each time during the 8-hour posttreatmentperiod, but mean values for TOBI 90 mg were less at each posttreatmenttime than those seen for TOBI 300 mg using the PARI LC PLUS nebulizer.

By 4 hours after the end of TOBI 30 mg and by 8 hours after TOBI 60 mgand 90 mg treatments, serum concentrations were below LOQ in at leasthalf of the patients [median (50^(th) percentile) serumconcentrations=0.0 μg/mL]. More than half of the TOBI 300 mg patientsremained above the serum LOQ at 8 hours posttreatment. There was noapparent pattern of change in posttreatment serum tobramycinconcentrations from period to period for any of the 4 treatments, andthere was no clear indication of the presence of a carryover (treatmentby period interaction) effect in posttreatment results.

Serum Pharmacokinetic Parameters

After TOBI administration using the Aerodose inhaler, mean C_(max) andAUC results increased linearly with dose after the administration of the30, 60, and 90 mg doses (Table 7), suggesting linear pharmacokinetics.Dose normalized AUC results were similar among the Aerodose dose levels,suggesting dose proportionality.

Comparing devices, C_(max) and AUC₀₋₈ for the TOBI 90 mg dose using theAerodose inhaler were not as high as results achieved by the TOBI 300 mgdose using the PARI LC PLUS nebulizer. However, the dose-normalizedparameters were higher for the Aerodose inhaler at all three TOBI doselevels, indicating better efficiency of the new device. Similar to thesputum data, the relative bioavailability was approximately 3-foldhigher for the Aerodose inhaler as compared to the PARI nebulizer. Thevariability based on AUCs was similar for both devices.

Exploratory analyses suggested that serum pharmacokinetic results wereunaffected by characteristics present before treatments began (age,gender, body weight, FEV₁% predicted at screening) and were unaffectedby events noted after the start of treatments (device failure,occurrence of bronchospasm defined as a decrease ≧10% in FEV₁, andrelative change in FEV₁% predicted).

TABLE 7 MEAN (SD) SERUM TOBRAMYCIN CONCENTRATIONS BY TIME ANDPHARMACOKINETIC PARAMETERS TOBI 300 mg Serum PARI LC TOBI 30 mg TOBI 60mg TOBI 90 mg Pharmacokinetic PLUS^(a) Aerodose inhaler^(b) Aerodoseinhaler^(b) Aerodose inhaler^(b) Parameters (n = 49) (n = 34) (n = 32)(n = 32) C_(max) (μg/mL) 1.12 (0.44) 0.38 (0.17) 0.69 (0.34) 0.96 (0.40)No. pts with data: 49 30 32 32 E vs C p-value^(c): <0.001 <0.001 0.027E/C (90% CIs)^(d): (0.29, 0.36) (0.53, 0.66) (0.75, 0.96)Dose-normalized C_(max) (μg/mL)/mg 0.0037 (0.0015) 0.0127 (0.0058)0.0116 (0.0056) 0.0106 (0.0045) No. pts with data: 49 30 32 32 E/C (90%CIs)^(d): (2.52, 3.25) T_(max) (hr) 1.05 (0.38) 1.14 (0.42) 0.98 (0.28)1.14 (0.64) No. pts with data: 49 30 32 32 T_(1/2) (hr) 3.42 (1.63) 6.75(5.31) 4.16 (2.34) 3.10 (1.10) Median T_(1/2) (hr) 3.14 4.27 3.42 2.73No. pts with data: 49 11 28 31 AUC₀₋₈ (hr · μg/mL) 4.96 (2.24) 1.43(1.43) 2.98 (1.92) 3.94 (1.52) No. pts with data: 49 30 32 32 E vs Cp-value^(c): <0.001 <0.001 0.165 E/C (90% CIs)^(d): (0.18, 0.25) (0.46,0.62) (0.75, 1.03) Dose-normalized AUC₀₋₈ 0.0166 (0.0075) 0.0478(0.0477) 0.0496 (0.0319) 0.0438 (0.0169) (hr · μg/mL)/mg No. pts withdata: 49 30 32 32 E/C (90% CIs)^(d): (2.51, 3.21) AUC_(0-∞) (hr · μg/mL)6.66 (4.32) 6.49 (7.71) 5.11 (4.62) 5.02 (1.63) No. pts with data: 49 1128 31 ^(a)Control (C) treatment = TOBI 300 mg delivered by PARI LC PLUSnebulizer. ^(b)Experimental (E) treatments = TOBI 30, 60, or 90 mgdelivered by Aerodose inhaler. ^(c)Pairwise contrast: TOBI 300 mg PARILC PLUS group vs TOBI (30, 60, 90 mg) Aerodose groups.^(d)Back-transformed 90% confidence intervals around the mean of the logratio of E and C treatments. Serum limit of quantifiability (LOQ): 0.2μg/mL.

Differences among treatment groups in serum C_(max) and AUC₀₋₈ (Table 7above; FIGS. 6 and 7) were statistically significant (p<0.001) with noperiod or carryover effects in the overall analyses. In pairwisecomparisons, C_(max) and AUC₀₋₈ were significantly greater for TOBI 300mg using the PARI LC PLUS than for TOBI 30 mg and TOBI 60 mg using theAerodose inhaler (p<0.001 in each comparison). C_(max) was statisticallysignificantly higher (p=0.027) for TOBI 300 mg compared to the TOBI 90mg dose, and AUC₀₋₈ was slightly but not significantly (p=0.165) greaterfor TOBI 300 mg than for TOBI 90 mg.

The Aerodose inhaler was more efficient, regardless of TOBI dose, thanthe PARI LC PLUS nebulizer based on dose normalized sputum C_(max) andAUC₀₋₈ results. Dose normalized means for these pharmacokineticparameters were similar among Aerodose treatments but approximately3-fold higher than the dose normalized results after TOBI 300 mgdelivered by the PARI LC PLUS nebulizer (Table 7).

T_(max) (Table 7) was similar for the four treatments, averaging between0.98 and 1.14 hours for Aerodose treatments and 1.05 hours for the TOBI300 mg treatment using the PARI LC PLUS. Median T_(1/2) ranged from 2.73to 4.27 hours among the Aerodose dose levels, compared to 3.14 hours forTOBI 300 mg using the PARI LC PLUS nebulizer. Median T_(1/2) resultsusing the Aerodose inhaler appeared to decrease with increasing TOBIdose, but this was considered an artifact related to greater frequencyof missing T_(1/2) values (due to more BQL results) at lower TOBI doselevels.

Exploratory analyses revealed no substantial association between serumpharmacokinetic results and patient characteristics present beforetreatments (age, gender, body weight, pulmonary function [FEV₁%predicted] at screening) or emergent events after the start oftreatments (device failure, occurrence of bronchospasm [decrease ≧10% inFEV₁ from predose to 30 minutes postdose], relative change in FEV₁.

Urinary Recovery of Tobramycin

Thirty-nine (39) of 49 completing patients had no measurable urinetobramycin concentrations before dosing in any of the 3 treatmentperiods, and 10 patients exhibited measurable predose urine tobramycinabove the lower LOQ in the periods indicated in Table 8 below.

TABLE 8 MEASURABLE TOBRAMYCIN IN PREDOSE URINE SPECIMENS Measurable^(b)Predose Previous Period Tobramycin during 8-24 hour Urine PeriodListed - Urine Tobramycin Serum Tobramycin Treatment^(a) TOBI doseConcentration T_(1/2) Concentration Patient Sequence (mg) (μg/mL) (hr)(μg/mL) 103-1005 C-1-2 prestudy na^(d) na^(d) Per 1 -- 3.80 300 3.924.80 Per 2 -- 2.06 30 2.48 not estimable Per 3 -- 1.20 103-1039 C-1-3prestudy na^(d) na^(d) Per 1 -- 1.82 300 6.76 1.87 Per 2 -- 2.58104-1024 C-1-3 300 5.14 3.16 Per 2 -- 1.48 107-1027 C-1-3 prestudyna^(d) na^(d) Per 1 -- 3.14 300 6.04 1.68 Per 2 -- 1.58 104-1020 C-2-1prestudy na^(d) na^(d) Per 1 -- 1.74 300 13.40 2.93 Per 2 -- 2.28 605.80 12.96 Per 3 -- 1.30 109-1014 C-2-3 60 <1.0 4.06 Per 3 -- 13.22106-1025 1-C-2 300 5.14 3.80 Per 3 -- 2.70 103-1012 2-C-3 300 2.26 3.63Per 3 -- 1.16 101-1002 3-C-1 300 7.82 3.37 Per 3^(c) -- 1.12 103-10063-C-2 prestudy na^(d) na^(d) Per 1 -- 2.72 90 10.10 3.14 Per 2 -- 3.10300 8.06 4.48 Per 3 -- 2.08 ^(a)Treatments: C = Control TOBI 300 mgusing PARI LC PLUS; 1 = TOBI 30 mg using Aerodose inhaler; 2 = TOBI 60mg using Aerodose inhaler; 3 = TOBI 90 mg using Aerodose inhaler.^(b)Measurable tobramycin in urine: tobramycin concentration > LOQ (1.0μg/mL). ^(c)Dosing interrupted by inhaler malfunction. ^(d)na = notapplicable; previous urine specimens were not collected.

Table 8 shows that measurable urine tobramycin was recovered beforedosing in periods 2, 3, or both for all 10 patients. Nine of the 10patients had measurable predose urine tobramycin after TOBI 300 mgtreatment during the preceding study period. One patient exhibitedmeasurable tobramycin in both predose serum and predose urine, and theseevents both followed TOBI 300 mg administration during the previousperiod.

Although carryover effect cannot be ruled out, the overall resultssuggest that such an effect is unlikely. The elimination half-life insputum ranged from 1.60 to 2.06 hours, and in serum ranged from 2.73 to4.27 hours, with no substantial differences between the four treatments.Additionally, the amount of tobramycin excreted in urine was largerduring the 0-8 hour period compared to the 8-24 hour period, consistentwith the short T_(1/2) of tobramycin. More importantly, in clinicalPhase III studies in patients, multiple daily administrations did notresult in any accumulation. Therefore it can be concluded that suchcarryover effect is most likely due to nonspecificity of the assay.

Consistent with the serum data, the amount of tobramycin excreted inurine was higher for TOBI 300 mg compared to TOBI 90 mg (Table 9 below).However, the percent of dose excreted in urine was 3-fold higher for theAerodose inhaler at all dose levels (16 to 18%) as compared to the PARILC PLUS nebulizer.

TABLE 9 MEAN (SD) URINARY RECOVERY OF TOBRAMYCIN BY TIME Urine TOBI 300mg TOBI 30 mg TOBI 60 mg TOBI 90 mg Tobramycin PARI LC PLUS^(a) Aerodoseinhaler^(b) Aerodose inhaler^(b) Aerodose inhaler^(b) Recovery (n = 49)(n = 34) (n = 32) (n = 32) Collection Interval Before and After Dosing:12-0 hr predose 305.1 122.8 67.9 615.5 (μg) (1412.0) (340.7) (192.8)(3202.5) No. pts with data 48 33 32 31 0-8 hr postdose 15003.0 4835.68490.3 12304.8 (μg) (7116.2) (2649.6) (3159.6) (5352.7) No. pts withdata 48 34 32 32 Dose-normalized 50.0 161.2 141.5 136.7 (μg)/mg (23.7)(88.3) (52.7) (59.5) No. pts with data 48 34 32 32 E/C (90% CIs)^(d):(2.50, 3.62) 8-24 hr postdose 3072.1 794.1 1367.4 2095.2 (μg) (2271.2)(853.1) (1118.8) (1818.7) No. pts with data 47 34 31 31 Dose-normalized10.2 26.5 22.8 23.3 (μg)/mg (7.6) (28.4) (18.6) (20.2) No. pts with data47 34 31 31 E/C (90% CIs)^(d): (2.44, 3.48) Total 0-24 hour 18113.25629.7 9802.7 14588.1 (μg) (8303.4) (2993.6) (3771.0) (6044.9) No. ptswith data 46 34 31 31 Dose-normalized 60.4 187.7 163.4 162.1 (μg)/mg(27.7) (99.8) (62.8) (67.2) No. pts with data 46 34 31 31 E/C (90%CIs)^(d): (2.23, 3.27) Percent of Dose 6.0 18.8 16.3 16.2 Excreted(%)^(c) ^(a)Control (C) treatment = TOBI 300 mg delivered by PARI LCPLUS nebulizer. ^(b)Experimental (E) treatments = TOBI 30, 60, or 90 mgdelivered by Aerodose inhaler. ^(c)% excreted = [(urinary recovery in μg÷ 1000 μg/mg) ÷ Dose in mg] · 100%. Urine limit of quantifiability(LOQ): 1.0 μg/mL urine.

For the Aerodose inhaler, mean 24-hour recovery of tobramycin from theurine increased with increasing TOBI dose during the study (Table 9above; FIG. 8). Tobramycin recovery appeared to be dose proportional forthe Aerodose inhaler, as mean 24-hour recovery normalized for dose wassimilar among Aerodose treatments.

Comparing devices, mean recovery for the TOBI 90 mg treatment was lessthan that seen for TOBI 300 mg using the PARI LC PLUS nebulizer.However, a greater percentage of the administered TOBI dose wasrecovered in the urine of patients who were dosed with the Aerodoseinhaler (18.8%, 16.3%, and 16.2%, respectively), irrespective of TOBIdose, than was recovered from patients who were dosed with the PARI LCPLUS nebulizer (6.0% of the administered TOBI 300 mg dose).

The largest amount of tobramycin was recovered during the first 8 hoursafter dosing. There was no apparent pattern of period-to-period changein posttreatment urine tobramycin recovery for any of the 4 treatments.Although a potential carryover could not be ruled out in approximately20% of the patients due to recovery of measurable tobramycin in predoseurine, there was no clear indication of the presence of a carryover(treatment by period interaction) effect in posttreatment results.

The percent of administered dose recovered in urine over 24 hourspostdose does not represent the delivered dose in the lung or absolutebioavailability. It is understood that a substantial amount of lungdeposited dose still remains in the body at 24 hours postdose.

Nebulization Time

Mean total nebulization time increased with increasing TOBI dose (Table10 below; FIG. 9) and was substantially less when the Aerodose inhalerwas used at each TOBI dose level (mean±SD for TOBI 30 mg=2.8±1.0 min;TOBI 60 mg=5.4±2.1 min; TOBI 90 mg=8.0±2.5 min) than when the PARI LCPLUS nebulizer was used (TOBI 300 mg=17.7±4.7 min).

TABLE 10 MEAN (SD) NEBULIZATION TIME TOBI 300 mg TOBI 30 mg TOBI 60 mgTOBI 90 mg PARI LC PLUS¹ Aerodose inhaler² Aerodose inhaler² Aerodoseinhaler² Parameter (n = 51) (n = 34) (n = 32) (n = 33) Nebulization 17.7(4.7) 2.8 (1.0) 5.2 (2.1) 8.0 (2.5) Time³ (min) No. pts with data 51 3432 32 ¹Control (C) treatment = TOBI 300 mg delivered by PARI LC PLUSnebulizer. ¹Experimental (E) treatments = TOBI 30, 60, or 90 mgdelivered by Aerodose inhaler. ²Total duration of nebulization excludingfill time.Conclusions

The Aerodose inhaler substantially reduced the amount of time requiredto nebulize the administered TOBI dose, compared to the approved PARI LCPLUS nebulizer, and nebulization time increased with increasing TOBIdose (TOBI 300 mg delivered by PARI LC PLUS mean=17.7 minutes vs. 2.8minutes, 5.4 minutes, and 8.0 minutes for TOBI 30 mg, 60 mg, and 90 mg,respectively).

Sputum tobramycin concentrations throughout the 8-hour sampling periodafter dosing increased with increasing TOBI dose through 90 mg deliveredby the Aerodose inhaler, but results for TOBI 90 mg and TOBI 300 mgdelivered by the PARI LC PLUS nebulizer did not differ substantially orconsistently. Sputum tobramycin results were highly variable, withcoefficients of variation approaching or exceeding 100% for eachtreatment at all time points. On average, sputum concentrations reachedtheir maximum at 10 minutes after each of the 4 treatments. By 2 hoursafter TOBI 30 mg and by 8 hours after TOBI 60 mg, 90 mg, and 300 mg,sputum concentrations were below the lower limit of quantifiability(LOQ) in at least half of the patients.

The mean of the maximum sputum concentration was significantly greaterafter TOBI 300 mg (mean=985.65 μg/gm) than after TOBI 30 mg (329.05μg/gm: p<0.001) and TOBI 60 mg (577.83 μg/gm: p=0.002) but not TOBI 90mg (958.00 μg/gm: p=0.856; 90% CIs for the ratio of TOBI 90 mg/TOBI 300mg C_(max)=0.72, 1.30). The Aerodose inhaler was more efficient than thePARI LC PLUS nebulizer based on sputum C_(max) results adjusted for TOBIdose administered (TOBI 300 mg with PARI LC PLUS: dose-normalized meanC_(max)=3.29 (μg/gm)/mg; TOBI 30, 60, and 90 mg with Aerodose=10.97,9.63, and 10.64 (μg/gm)/mg, respectively).

Mean sputum T_(max) was virtually identical for TOBI 300 mg (mean=0.26hr) and TOBI 30 mg (0.24 hr) but was slightly less than T_(max) for TOBI60 mg (0.38 hr) and TOBI 90 mg (0.33 hr).

Mean sputum AUC₀₋₈ was significantly greater after TOBI 300 mg(mean=1471.16 hr•μg/gm) than after TOBI 30 mg (360.79 hr•μg/gm: p<0.001)and TOBI 60 mg (804.78 hr•μg/gm: p<0.001) but not TOBI 90 mg (1275.23hr•μg/gm: p=0.465; 90% CIs for the ratio of TOBI 90 mg/TOBI 300 mgAUC₀₋₈=0.72, 1.14). The greater efficiency of the Aerodose inhaler wasalso seen in dose-normalized AUC₀₋₈ results (TOBI 300 mg with PARI LCPLUS=4.90 [hr•μg/gm]/mg; TOBI 30, 60, and 90 mg with Aerodose=12.03,13.41, and 14.17 [hr•μg/gm]/mg, respectively).

No inferential analyses of sputum AUC_(0-∞) were performed due to highvariability that increased with increasing TOBI dose.

Serum tobramycin concentrations also increased with increasing TOBI doseat each time during the 8-hour posttreatment observation period. Meanserum tobramycin concentrations reached their maximum at one hour aftereach treatment. By 4 hours after TOBI 30 mg and by 8 hours after TOBI 60mg and TOBI 90 mg, serum concentrations were below LOQ in at least halfof the patients. More than half of the TOBI 300 mg patients remainedabove the serum LOQ at 8 hours posttreatment.

Mean serum C_(max) was significantly greater after TOBI 300 mg(mean=1.12 μg/mL) than after the other 3 treatments (TOBI 30 mg=0.38μg/mL, p<0.001; TOBI 60 mg=0.69 μg/mL, p<0.001; TOBI 90 mg=0.96 μg/mL,p=0.027). The Aerodose inhaler was also more efficient than the PARI LCPLUS nebulizer based on serum C_(max) results adjusted for TOBI doseadministered (TOBI 300 mg with PARI LC PLUS: dose-normalized meanC_(max)=0.0037 (μg/mL)/mg; TOBI 30, 60, and 90 mg with Aerodose=0.0127,0.0116, and 0.0106 (μg/mL)/mg, respectively.

Mean serum T_(max) was similar for the 4 treatments (mean=1.05 hr, 1.02hr, 0.98 hr, and 1.14 hr for TOBI 300 mg, 30 mg, 60 mg, and 90 mg,respectively).

Mean serum AUC₀₋₈ was significantly greater after TOBI 300 mg (mean=4.96hr•μg/mL) than after TOBI 30 mg (1.43 hr•μg/mL, p<0.001) and TOBI 60 mg(2.98 hr•μg/mL, p<0.001) but not TOBI 90 mg (3.94 hr•μg/mL, p=0.165; 90%CIs for the ratio of TOBI 90 mg/TOBI 300 mg AUC₀₋₈=0.75, 1.03). Thegreater efficiency of the Aerodose inhaler was also seen indose-normalized AUC₀₋₈ results (TOBI 300 mg with PARI LC PLUS=0.0166[hr•μg/mL]/mg; TOBI 30, 60, and 90 mg with Aerodose=0.0478, 0.0496, and0.0438 [hr•μg/mL]/mg, respectively).

Serum AUC_((0-∞)) was not analyzed statistically due to high variabilitybut generally appeared to increase as the TOBI dose increased.

Recovery of tobramycin from the urine within 24 hours after dosingincreased with increasing TOBI dose during the study (expressed in mg[mg=μg/1000], mean urine tobramycin recovery=18.1 mg, 5.6 mg, 9.8 mg,and 14.6 mg after TOBI 300 mg, TOBI 30 mg, TOBI 60 mg, and TOBI 90 mgdoses, respectively). Most of the tobramycin was recovered within thefirst 8 hours after dosing. Normalized for dose, urine tobramycinrecovery within 24 hours was 6.0%, 18.8%, 16.3%, and 16.2% of theadministered TOBI 300 mg, TOBI 30 mg, TOBI 60 mg, and TOBI 90 mg doses,respectively.

Results of the present study showed that TOBI 300 mg delivered by thePARI LC PLUS nebulizer (the control delivery system) and TOBI 30 mg, 60mg, and 90 mg delivered by the Aerodose inhaler (the experimentaldelivery system) were safe and well-tolerated by male and female cysticfibrosis patients. Fifteen patients (9 male and 6 female) experienced 24instances of bronchospasm (decline in FEV₁ (liters)≧10%). There were nostatistically significant differences between control and anyexperimental treatment in the incidence of bronchospasm. There were nooverall treatment differences in quantitative change in FEV₁ frompredose to 30-minute postdose measurement times.

The study found no evidence that CF patients were at increased risk byreason of inhaling single TOBI doses of 30 mg, 60 mg or 90 mg comparedto the single TOBI 300 mg dose delivered by the PARI LC PLUS jetnebulizer. The most frequently reported treatment emergent adverseevents (cough increased, rhinitis, sputum increased, chest pain, asthma,and headache) and the SAEs reported by 4 of the patients were primarilyassociated with patients' underlying CF disease and related medicalconditions. The incidence of these events before and after studytreatments was substantially similar, suggesting that neither TOBI doselevels nor control and experimental inhalers altered ongoingsymptomatology associated with patients' underlying medical conditions.There were also no clinically significant safety issues reflected inclinical laboratory test results, vital signs, or physical findings.

In this example, the Aerodose inhaler substantially reduced the timerequired for nebulization of all three dose levels (30 mg, 60 mg, and 90mg) of TOBI compared to the nebulization time for the approved TOBI 300mg delivery system using the PARI LC PLUS jet nebulizer. Averagenebulization times were 2.8, 5.4, and 8.0 minutes using the Aerodoseinhaler to deliver TOBI 30 mg, 60 mg, and 90 mg, respectively vs. 17.7minutes using the PARI LC PLUS nebulizer to deliver TOBI 300 mg. TheAerodose inhaler therefore cut nebulization time of the TOBI 90 mg doseby more than 50% compared to the PARI LC PLUS nebulizer in the presentstudy, and nebulization times for lower TOBI doses were reduced by evengreater amounts. Present nebulization time results in CF patients ≧12years of age with baseline FEV₁% predicted ≧40% were consistent withthose obtained after single doses of TOBI 60 mg using the Aerodoseinhaler (mean=5.7 minutes) but slightly less than TOBI 300 mg resultsusing the PARI LC PLUS nebulizer (mean=20.4 minutes) in the TOBI gammascintigraphy study of tobramycin deposition in the lungs of healthyadult male and female volunteers of Example 2, infra.

This example demonstrates that TOBI 90 mg (but not TOBI 60 mg or TOBI 30mg) delivered by the Aerodose inhaler achieved similar actual pulmonarydeposition, systemic absorption, and urinary recovery of tobramycin asthat achieved by administration of the TOBI 300 mg dose delivered by thePARI LC PLUS nebulizer. Normalized for TOBI dose, the Aerodose inhalerwas substantially more efficient than the PARI LC PLUS nebulizer in thedelivery of aerosolized tobramycin to the lungs and to the systemiccirculation.

Pulmonary deposition of tobramycin was measured by determination ofsputum tobramycin concentrations and by calculation of sputumpharmacokinetic parameters. Maximum sputum tobramycin concentrationswere reached by 10 minutes after administration of each treatment, andconcentrations were below the LOQ in half or more of the patients at 2hours after TOBI 30 mg and at 8 hours after TOBI 60 mg, 90 mg, and 300mg. The extent of pulmonary deposition of tobramycin, as measured bymaximum sputum concentrations and sputum AUC₀₋₈ results, increased withincreasing TOBI dose through 90 mg, but TOBI 90 mg and TOBI 300 mg didnot differ statistically (mean sputum C_(max)=958.00 and 985.65 μg/gm;mean sputum AUC₀₋₈=1275.23 and 1471.16 hr•μg/gm, respectively). Meansputum C_(max) results after TOBI 30 mg and 60 mg doses weresignificantly less than that of the TOBI 300 mg dose. Present sputumC_(max) results achieved after the single TOBI 300 mg dose were slightlyless than sputum tobramycin concentrations achieved 10 minutes after asingle TOBI 300 mg dose (mean sputum tobramycin concentration=1237μg/gm, median=1090 μg/gm) in two large previously conducted Phase IIIpivotal trials.

The results of this example demonstrate that at least one of the threeTOBI doses (TOBI 90 mg) delivered by the experimental Aerodose inhalerachieved similar actual sputum tobramycin concentrations and that theseresults in turn were similar to sputum results obtained in the priorpivotal studies supporting the commercial TOBI product. It is alsoimportant that present sputum results demonstrated that the experimentalAerodose inhaler was substantially more efficient, regardless of TOBIdose, in delivery of aerosolized tobramycin to the lung than the PARI LCPLUS jet nebulizer. Dose-normalized sputum C_(max) was 10.97, 9.63, and10.64 (μg/gm)/mg for TOBI 30 mg, 60 mg, and 90 mg delivered by Aerodoseinhaler, respectively, compared to 3.29 (μg/gm)/mg for TOBI 300 mgdelivered by PARI LC PLUS. Similarly, dose-normalized sputum AUC₀₋₈ was12.03, 13,41, and 14.17 [hr•μg/gm]/mg for TOBI 30 mg, 60 mg, and 90 mgdelivered by Aerodose inhaler, respectively, compared to 4.90[hr•μg/gm]/mg for TOBI 300 mg delivered by PARI LC PLUS.

Systemic absorption of tobramycin was measured by determination of serumtobramycin concentrations and by calculation of serum pharmacokineticparameters. Maximum serum tobramycin concentrations were reached at onehour after each of the four TOBI treatments, and concentrations werebelow LOQ in half or more of the patients by 4 hours after TOBI 30 mgand by 8 hours after TOBI 60 mg and 90 mg. More than half of thepatients at TOBI 300 mg had measurable serum tobramycin at 8 hourspostdose. The extent of absorption of tobramycin, as measured by serumC_(max) results, increased with increasing TOBI dose, as C_(max) wassignificantly greater after TOBI 300 mg (mean=1.12 μg/mL) than aftereach of the lower TOBI doses (mean=0.38, 0.69, and 0.96 μg/mL for TOBI30 mg, 60 mg, and 90 mg doses, respectively). Serum C_(max) for TOBI 300mg in the present study was slightly higher (mean±SD=1.10±0.44 μg/mLwith a mean T_(max) of 1.05 hr) than the mean serum tobramycinconcentration reported at one hour after TOBI 300 mg in the TOBI NDA(0.95±0.50 μg/mL). Serum C_(max) achieved by the Aerodose inhaler at theTOBI 90 mg dose level in the current study was virtually identical tothe NDA serum concentrations one hour after TOBI 300 mg (mean=0.96±0.37μg/mL), although it was significantly (p=0.027) less than the currentTOBI 300 mg.

Thus, present serum tobramycin results demonstrated that TOBI 90 mgdelivered by the Aerodose inhaler were similar (AUC₀₋₈) or nearlysimilar (C_(max)) to those obtained after TOBI 300 mg delivered by thePARI LC PLUS nebulizer in the present study and in the prior pivotalstudies supporting the TOBI commercial product. Present serum resultsalso demonstrated that the experimental Aerodose inhaler wassubstantially more efficient, regardless of TOBI dose, in delivery ofaerosolized tobramycin to the systemic circulation than the PARI LC PLUSjet nebulizer. Dose-normalized serum C_(max) was 0.0127, 0.0116, and0.0106 (μg/mL)/mg for TOBI 30 mg, 60 mg, and 90 mg delivered by Aerodoseinhaler, respectively, compared to 0.0037 (μg/mL)/mg for TOBI 300 mgdelivered by PARI LC PLUS. Similarly, dose-normalized serum AUC₀₋₈ was0.0478, 0.0496, and 0.0438 [hr•μg/mL]/mg for TOBI 30 mg, 60 mg, and 90mg delivered by Aerodose inhaler, respectively, compared to 0.0166[hr•μg/mL]/mg for TOBI 300 mg delivered by PARI LC PLUS. The greaterefficiency of the Aerodose inhaler observed in present serum tobramycinresults is consistent with greater efficiency and less wastage of thetobramycin dose observed in earlier studies.

Urinary recovery of tobramycin was measured by determining thecumulative amount of tobramycin recovered in urine collected for 24hours after dosing. The amount of urinary tobramycin recovered within 24hours postdose increased with increasing TOBI dose (expressed in mg[mg=μg/1000], mean urine tobramycin recovery=5.6 mg, 9.8 mg, 14.6 mg,and 18.1 mg tobramycin after TOBI 30 mg, 60 mg, 90 mg, and 300 mg). Theresults were not tested statistically, and it was not possible todetermine whether TOBI 90 mg and TOBI 300 mg results for 24-hourrecovery of urine tobramycin were similar or different.

Normalized for dose by dividing the mean amount of tobramycin recoveredby the nominal amount of TOBI administered, urinary recovery oftobramycin was approximately 18.8%, 16.3%, 16.2%, and 6.0% of theadministered TOBI 30 mg, 60 mg, 90 mg, and 300 mg doses, respectively.

During the study, measurable tobramycin (i.e., above the lower limit ofquantifiability [LOQ] of the assay) was detected in 12-hour predoseurine collections in a total of 10 patients, including 5 patients beforethe first dose of study treatments in period one and all 10 patientsbefore the second or third doses in periods 2 and 3 or both. Similarly,measurable tobramycin was detected in predose serum specimens in a totalof 5 patients, including 3 patients before the first dose of studytreatments in period one and 4 patients before dosing in periods 2, or3, or both. A single patient had measurable tobramycin in both urine andserum.

Substantial variability is known to occur among patients in the rate andextent of uptake, renal accumulation, and elimination of aminoglycosideantibiotics, even in patients with normal renal function. Each of thesefactors may lengthen the amount of time that measurable concentrationsof aminoglycoside antibiotics may be detected in serum and urine. Thepresent study employed a prestudy washout interval of 7 days fromprevious prescription aminoglycoside antibiotic use and a 7-day intervalbetween the 3 single doses of TOBI, an aminoglycoside antibiotic, duringthe crossover treatment periods. It is plausible that prestudy andon-study washout intervals in the study may have been too short forcomplete elimination of residual tobramycin previously administered, ifany. Measurable amounts of tobramycin for these patients would have hadlittle effect on study results, since the amounts and concentrationsdetected were very small in nearly all cases, and no unusually highserum or urine tobramycin results were noted during the study.

The Aerodose inhaler was a safe and efficient aerosolization anddelivery device for TOBI during the study.

Example 2 Scintigraphy Study

In order to assess the in vivo lung deposition of 300 mg tobramycin(TOBI®) inhaled using the PARI LC PLUS™ jet nebulizer/DeVilbissPulmoAide® compressor delivery system (current commercial deliverysystem) compared with the deposition of 60 mg tobramycin (TOBI®) usingthe AeroDose™ inhaler in accordance with the present invention, a gammascintigraphy study was performed. The imaging technique of gammascintigraphy is a well-established method¹⁰⁻¹² that provides precisequantification of drug delivered to the lungs¹³. It also provides anassessment of the distribution of deposited drug in different lungregions (peripheral, intermediate and central lung regions correspondingto small airways, medium sized airways and large airways,respectively¹⁴). Gamma scintigraphy is the only non-invasive methodcurrently available for obtaining this type of information.

The study of this example was designed as an open label, randomized,single center, single dose, two period crossover Phase I study ofaerosol delivery characteristics and safety of two inhalation devices inhealthy adult volunteers. A maximum of 14 healthy male or non-pregnant,non breast-feeding female volunteers aged 18 to 65 years of age were toreceive in random order two single doses of aerosolized antibiotic mixedwith a sterile radiotracer (technetium bound todiethylenetriaminepentaacetic acid: ^(99m)Tc DTPA) separated by awashout interval of a minimum of 44 hours between doses. Radiolabeledaerosols consisted of a single 300 mg dose in a 5 mL solution of TOBIdelivered by the control delivery system (PARI LC PLUS jet nebulizerwith a PulmoAide compressor) and a single 60 mg dose in a 1 mL solutionof TOBI delivered by the experimental delivery system (Aerodoseinhaler).

Aerosol delivery characteristics of control and experimental deliverysystems were compared on the basis of lung deposition of radiolabeledtobramycin determined by gamma scintigraphy, time to completenebulization of aerosolized doses, serum concentrations of tobramycindetermined by Abbott TDxFLx assays, and serum tobramycin pharmacokineticparameters.

The safety of control and experimental TOBI delivery systems wascompared on the basis of changes in pulmonary function, the incidence oftreatment emergent adverse events, and the occurrence of clinicallysignificant laboratory and clinical evaluations and of unusually highserum tobramycin concentrations.

The duration of study participation for each subject was to beapproximately five weeks including a screening period of up to 3 weeksin duration, two treatment periods of approximately 9 hours eachseparated by a minimum 44-hour washout interval, and a follow-up periodthrough 2 weeks after the end of dosing.

Treatments

TOBI® was administered by inhalation as a single 300 mg dose and as asingle 60 mg dose to each subject during the study. The 300 mg dose wassupplied as a commercial ampoule of TOBI. The 60 mg dose of tobramycinsolution was prepared by study site personnel by withdrawing 1.0 mL ofsolution from the 300 mg/5 mL commercial ampoule of TOBI into two unitdose syringes containing 0.5 mL 30 each.

Sterile ^(99m)Tc DTPA was added as a radiotracer to both 300 mg and 60mg solutions at the study site prior to instillation into the nebulizer.Sufficient ^(99m)Tc DTPA was added to both the 300 mg and the 60 mg doseso that no more than 10 MBq ^(99m)Tc DTPA was delivered to the subjectwith each single dose administered.

Using control and experimental aerosol delivery systems, each subjectwas to self-administer two single aerosolized doses of radiolabeled(^(99m)Tc DTPA) TOBI, one dose in each of two crossover treatmentperiods, according to the randomization scheme for the study. Subjectswere instructed to use nose clips and breathe in a normal breathingpattern while inhaling the medication according to the instructions foruse for each inhaler.

Control and experimental treatment delivery systems were specified asfollows.

Control Treatment Delivery System: PARI LC PLUS jet nebulizer withDeVilbiss PulmoAide compressor delivering 300 mg (5 mL) of TOBI.

Experimental Treatment Delivery System: Aerodose inhaler delivering 60mg (1 mL) of TOBI.

When the PARI LC PLUS nebulizer was used, 5 mL radiolabeled TOBI wasadded to the drug reservoir and nebulized without interruption until thenebulizer reservoir was dry. The PARI system was configured such thatexhalation by the subject did not result in escape of radioactiveaerosol into the surrounding atmosphere. Exhaled droplets were collectedusing a filter attached to the side of the inhaler by a T-piece. Inaddition, a scavenger filter was placed above the inhaler, which was inturn connected to a vacuum pump. The scavenger system was used tocollect any radiolabeled droplets escaping from the inhaler.

When the Aerodose inhaler was used, one 0.5 mL aliquot of radiolabeledTOBI was added to the drug reservoir and nebulized to dryness. A second0.5 mL dose was then added to the reservoir and nebulized to dryness.The inhaler was surrounded with an exhaled air collection box. Air wasdrawn through a filter at the back of the box using a vacuum pump.

Start and stop times of nebulization for both the Aerodose and PARI LCPLUS nebulizers were to be recorded in CRFs. Nebulization time for theAerodose inhaler was not to include the time needed to refill the drugreservoir according to the protocol.

Enrolled volunteers were randomly assigned to two treatment sequencegroups as illustrated below according to a randomization scheme.

PARI 300 mg/Aerodose 60 mg:

-   -   period 1: PARI LC PLUS with TOBI 300 mg    -   period 2: Aerodose with TOBI 60 mg        Aerodose 60 mg/PARI 300 mg:    -   period 1: Aerodose with TOBI 60 mg    -   period 2: PARI LC PLUS with TOBI 300 mg

All subjects randomly assigned to a single treatment sequence groupreceived control and experimental treatments in the same order duringthe study, while subjects assigned to the other treatment sequence groupreceived treatments in the reverse order. Table 11 below shows the twosequences of treatment administration employed during the study via therandomization process.

TABLE 11 TREATMENT SEQUENCE GROUPS AND SEQUENCE OF TREATMENTS IN THESTUDY Treatment Sequence Treatment Treatment Group¹ Period 1 Period 2C-E² C E E-C E C ¹Subjects were randomly assigned to the two treatmentsequence groups. ²C and E refer to control and experimental treatmentsadministered during the study as follows: C = PARI LC PLUS jet nebulizer(60 mg/mL; 300 mg in 5 mL) E = Aerodose inhaler (60 mg/mL; 60 mg in 1.0mL)

Before dosing, TOBI formulations were radiolabeled with ^(99m)Tc-DTPA inpreparation for gamma scintigraphy to determine posttreatment tobramycindeposition in the lungs. Subjects practiced the inhalation procedurewith both control and experimental devices filled with normal saline.When the investigator was satisfied that the subject could reproduciblyperform the correct inhalation technique, the inhaler was filled withthe radiolabeled formulation, and the subject inhaled the radiolabeleddose until the nebulizer was dry and nebulization was stopped.

Immediately following inhalation of radiolabeled, aerosolizedtobramycin, scintigraphic images were recorded to determineradioactivity associated with lung and oropharyngeal tobramycindeposition and with external items such as nebulizer parts, mouthpieces,filters, and tissues used by subjects. If not previously done within thelast 5 years, a posterior lung ventilation scan was also performedduring the study after subjects inhaled the radioactive inert gas^(81m)Kr to determine the lung outlines and facilitate the determinationof regional deposition of radiolabeled tobramycin.

Deposition of Tobramycin

Assessment and comparison of tobramycin deposition patterns between PARILC PLUS and Aerodose delivery systems was a primary objective of thestudy. Deposition patterns of inhaled, radiolabeled tobramycin weredetermined using scintigraphic imaging methodology. Lung, oropharyngeal,and (if necessary) abdominal radioactivity was measured from imagesobtained immediately after inhalation of each single dose ofradiolabeled tobramycin using a gamma camera (General ElectricMaxicamera) with a 40 cm field of view and fitted with a low energyparallel hole collimator. Images were obtained as described below:

-   -   posterior view of the chest;    -   anterior view of the chest;    -   right lateral view of the oropharynx;    -   anterior and posterior abdominal views if necessary, i.e., if        activity had spread through the intestine, beyond the field of        view in either of the chest images;    -   items external to the body of the subject as follows:        -   for the PARI LC PLUS system:            -   nebulizer cup            -   mouthpiece            -   exhalation filter and T-piece            -   scavenger filter            -   any tissues used by the subject        -   for the Aerodose system:            -   Aerodose inhaler            -   exhaled air containment box and filter            -   any tissues used by the volunteer

Additionally, a posterior lung ventilation scan was performed using theradioactive inert gas, krypton (^(81m)Kr), to determine the lungoutlines. The lung outlines were used to divide lung images of eachsubject into central, intermediate, and peripheral lung zones in orderto determine the amount of aerosolized tobramycin deposited in each ofthese zones¹⁷. Lung ventilation scans taken for subjects whoparticipated in earlier studies were acceptable for use for this studyprovided the scan was obtained within the last five years and thesubject had no record of serious lung disease in the intervening period.

Deposition zones of interest on scintigraphic images were additionallydrawn around the oropharynx, esophagus, and stomach (including anyactivity in the small intestine). The counts obtained within all regionsof interest were corrected for background radioactivity, radioactivedecay, and for tissue attenuation¹⁸. In regions where both anterior andposterior images were recorded, the geometric mean of counts in bothimages was calculated prior to correction for tissue attenuation.Determination of the percentage of the dose deposited in the oropharynxincluded activity adhering to the mouth and oropharynx together with anyswallowed activity detected in the esophagus, stomach, and intestine.

All images were recorded using Micas X plus software installed on a UNIXbased computer system. Images were stored on digital audio tape (DAT)for subsequent analysis and archiving. Scintigraphic data were analyzedby Pharmaceutical Profiles Ltd. (PPL) in accordance with the PPLStandard Operating Procedure N 1013 “Lung Quantitative Data Analysis”.The data were summarized to obtain the following parameters:

-   -   whole lung deposition (% of metered dose);    -   central lung zone deposition (% of metered dose);    -   intermediate lung zone deposition (% of metered dose);    -   peripheral lung zone deposition (% of metered dose);    -   ratio of peripheral to central zone deposition (lung penetration        index);    -   oropharyngeal deposition (including esophagus and stomach) (% of        metered dose);    -   inhaler deposition (PARI LC PLUS or AeroDose) (% of metered        dose);    -   radioaerosol in exhaled air (filters) (% of metered dose);    -   radioaerosol on PARI LC PLUS mouthpiece, T-piece, scavenger        filter and subject tissues (% of metered dose);    -   radioaerosol on Aerodose exhaled air collection box and subject        tissues (% of metered dose).

The counts in each area were expressed as a percentage of the metereddose that was determined from the sum of the total body counts inaddition to those deposited on the inhaler and the exhalation filter.Since the volume of TOBI placed into each of the two inhalers wasdifferent, direct comparisons of the percentage deposition values wasproblematic. To aid interpretation of the data, the percentagedeposition values were multiplied by the nominal metered dose (300 mgfor the PARI LC PLUS and 60 mg for the Aerodose inhaler) to obtainamounts of drug deposited in milligrams for each of the depositionparameters listed above.

Nebulization Time

Assessment and comparison of nebulization time between PARI LC PLUS andAerodose delivery systems was another objective of the study. Elapsedtime from the start of nebulization (defined as the subject's firsttidal breath after the inhaler was in place) until no more TOBI solutionwas aerosolized by the inhaler was measured by staff at the site using astopwatch. Nebulization time was not to include time needed forinstillation of drug into the nebulizer between the repeat filling ofthe Aerodose inhaler. The length of any interruption in nebulization andthe reason for interruption were recorded.

Serum tobramycin concentrations were determined for the present study,and pharmacokinetic parameters were calculated, to provide preliminaryestimates of the bioavailability of 60 mg TOBI delivered by the Aerodosesystem in comparison with that of the marketed 300 mg TOBI formulation.Additionally, unusually high serum tobramycin results (≧4 μg/mL) wereconsidered an important measure of safety during the study.

Venous blood samples (8 mL) for the determination of serum tobramycinconcentrations were collected by intravenous cannula or by venipuncturebefore each single dose of TOBI and at 30 minutes and 1, 2, 4, and 8hours after completion of dosing. The first one mL of blood withdrawnfrom the cannula was discarded, and the subsequent 7 mL was withdrawninto serum sampling tubes. Cannulae were frequently flushed with salineduring the course of the treatment day.

Blood samples were centrifuged at approximately 1600 g for 10 minutes at4° C. The resulting serum fraction was split into two aliquots bypipetting into two prelabeled polypropylene screw cap tubes. Tubes werestored at −20° C. for each study period and were then transferred to a−70° C. freezer.

The maximum tobramycin concentration (C_(max)) and the time to reachC_(max) (T_(max)) were the observed values. The elimination rateconstant (k_(el:) used to calculate AUC_(0-∞); see next paragraph) wascalculated as the negative slope of the log plasma concentration vs.time plot using the last two measurable concentrations. Use of more thantwo concentrations at or after T_(max) is preferred for calculation ofthe elimination rate constant; however, several subjects had only twomeasurable tobramycin concentrations at the terminal phase after TOBI 60mg using the Aerodose inhaler. The alternate method of calculatingk_(el) using the last two measurable concentrations was employed for allsubjects for both period 1 and period 2.

Area under the curve through 8 hours postdose (AUC₀₋₈) and extrapolatedto infinity (AUC_(0-∞)) were calculated for serum tobramycinconcentrations using the linear trapezoid rule. Actual nebulization timewas added to the time between predose and 30 minutes after the end ofinhalation when calculating AUC₀₋₈. AUC_(0-∞) was extrapolated from thelast measurable concentration to infinite time by adding the quantityequal to the last measurable concentration divided by the eliminationrate constant (k_(el)).

Statistical Methods Planned in the Protocol

Scintigraphic data were analyzed in accordance with the current versionof the PPL Standard Operating Procedure N 1013 “Lung Quantitative DataAnalysis”. Manipulation and calculation of radioactivity counts wereaccomplished using a custom written region of interest program, whereregions of interest were central, intermediate, peripheral, andstomach/intestines if necessary. Numerical data were downloadedautomatically from the Park Medical computer into a customizedspreadsheet.

Due to the small number of subjects in the study, statistical analysiswas performed only on whole lung deposition data and on selectedpharmacokinetic parameters. All other study data were summarizeddescriptively. Descriptive summaries for quantitative data includedsample size, mean, standard deviation, median, minimum, maximum, and/orrange values as appropriate. Descriptive summaries for qualitative orcategorical data included number and percent of subjects with thecharacteristic. All clinical data manipulations, analyses, summaries,and transformations employed SAS version 6.12²⁰⁻²².

Aerosol Delivery Analyses

Whole lung deposition was the primary endpoint for the analysis. TheWilcoxon one-sample, matched-pairs, signed ranks test was used todetermine whether differences between the whole lung deposition patterns(percent and amount of metered dose deposited) for the two inhalers weresignificant. The significance level was set at α=0.05.

Serum pharmacokinetic parameters (C_(max), AUC₀₋₈, and AUC_(0-∞)) wereanalyzed for differences between delivery systems using a repeatedmeasures analysis of variance. The statistical model included studyperiod and delivery systems as fixed effects and subject as the randomeffect. The carryover effect from treatment period 1 to 2 was alsoinvestigated. The significance level was set at α=0.05, and tests ofsignificance were two-sided.

Additional deposition measures of interest, nebulization time, serumtobramycin concentrations and pharmacokinetic parameters were summarizedand evaluated descriptively for apparent differences between aerosoldelivery systems.

Study Drug Administration

All subjects were successfully dosed according to the randomizationschedule for the study, and all subjects received and completed bothinhalation administrations. All subjects received single doses of TOBI300 mg and TOBI 60 mg during the study.

Deposition of Radiolabeled Tobramycin

Tobramycin deposition results indicated that the Aerodose system wasmore efficient than the PARI LC PLUS system. The Aerodose system withTOBI 60 mg delivered a greater percentage of the dose to the lungs(mean±SD=34.8±10.1%) than the PARI system with TOBI 300 mg (8.2±3.6%),and the difference was statistically significant (p=0.005) (see Table 12below). Results from the analysis (n=9) that excluded data from onepatient were similar (means=35.4% vs. 9.1% for Aerodose and PARIsystems, respectively; p=0.008).

The actual amount of drug delivered to the lungs (Table 13 below) wasslightly but not significantly less (p=0.202) using the Aerodose inhaler(20.9±6.0 mg) than using the PARI inhaler (24.5±10.7 mg). Excludingsubject 1007, the analysis showed significantly less (p=0.04) depositionof the Aerodose 60 mg dose (21.2 mg) than the PARI 300 mg dose (27.2mg).

TABLE 12 MEAN (SD) PERCENTAGE DEPOSITION OF THE METERED TOBI DOSE Intentto Treat Excluding Subject 1007 (n = 10) (n = 9) TOBI TOBI 300 mg TOBI300 mg TOBI Zone of PARI LC 60 mg PARI LC 60 mg Deposition PLUS AeroDosePLUS AeroDose p-value* Whole lung  8.2 (3.6)*  34.8 (10.1)* 9.1 (2.2) 35.4 (10.5) 0.005 central 2.4 (1.2) 10.1 (4.0) 2.7 (0.9) 10.2 (4.2)intermediate 2.7 (1.2) 11.6 (3.6) 3.0 (0.8) 11.8 (3.7) peripheral 3.1(1.3) 13.2 (3.4) 3.5 (0.7) 13.4 (3.5) ratio: peripheral/central 1.2(0.5)  1.4 (0.4) 1.4 (0.3)  1.4 (0.4) Oropharynx (including 14.4 (6.7)  31.5 (11.6) 16.0 (4.7)   31.5 (12.3) esophagus and stomach) Inhaler42.6 (6.7)  15.2 (8.4) 43.5 (6.4)  15.1 (8.9) Exhalation filter 31.6(10.9) 16.9 (5.6) 28.3 (2.7)  16.3 (5.6) PARI-specific: mouthpiece 1.0(0.5) 1.0 (0.5) T-piece 2.0 (0.6) 2.0 (0.5) tissue 0.0 (0.1) 0.0 (0.1)scavenger filter 0.1 (0.1) 0.1 (0.1) AeroDose-specific: box  1.7 (1.5) 1.6 (1.6) tissue  0.0 (0.1)  0.0 (0.1) *Wilcoxon matched-pairs signedranks test on intent to treat dataset. Excluding Subject 1007: p =0.008. Statistical significance: p ≦ 0.05.

The Aerodose inhaler deposited proportionally more (Table 12 above)tobramycin in the lungs than in the oropharynx (mean 34.8% vs. 31.5% ofthe 60 mg dose), while the PARI LC PLUS nebulizer deposited lesstobramycin in the lungs than in the oropharynx (mean 8.2% vs. 14.4% ofthe 300 mg dose). The ratio of lung to oropharyngeal deposition (wholelung deposition divided by oropharynx deposition in Table 12 above) wasapproximately 1.1 for the Aerodose inhaler compared to approximately 0.6for the PARI LC PLUS nebulizer.

Regional deposition within the lung was predominantly peripheral andvery similar for both inhalers (ratio of radioactivity in peripheral tocentral zones: Aerodose=1.4±0.4; PARI LC PLUS=1.2±0.5).

Substantially less tobramycin was deposited on the Aerodose inhaler(15.2±8.4%; 9.1±5.1 mg; Tables 4 and 5, respectively) and exhalationfilter (16.9±5.6%; 10.1±3.3 mg) than on the PARI LC PLUS nebulizer(42.6±6.7%; 127.8±20.0 mg) and filter (31.6±10.9%; 94.8±32.7 mg). Nomore than 2% of the metered dose was deposited on inhaler-specificsurfaces or tissue paper used by subjects.

TABLE 13 MEAN (SD) AMOUNT (MG) OF DEPOSITION OF THE METERED TOBI DOSEIntent to Treat Excluding Subject 1007 (n = 10) (n = 9) TOBI TOBI 300 mgTOBI 300 mg TOBI Zone of PARI LC 60 mg PARI LC 60 mg Deposition PLUSAeroDose PLUS AeroDose p-value* Whole lung  24.5 (10.7)* 20.9 (6.0)*27.2 (6.7)  21.2 (6.3)  0.202 central 7.3 (3.6) 6.0 (2.4) 8.0 (2.8) 6.1(2.5) intermediate 8.0 (3.7) 6.9 (2.1) 8.9 (2.5) 7.1 (2.2) peripheral9.3 (3.8) 7.9 (2.1) 10.4 (2.0)  8.1 (2.1) Oropharynx (including 43.3(20.2) 18.9 (6.9)  48.1 (14.0) 18.9 (7.4)  esophagus and stomach)Inhaler 127.8 (20.0)  9.1 (5.1) 130.5 (19.2)  9.0 (5.4) Exhalationfilter 94.8 (32.7) 10.1 (3.3)  84.8 (8.1)  9.8 (3.4) PARI-specific:mouthpiece 3.0 (1.4) 3.1 (1.5) T-piece 6.1 (1.7) 5.9 (1.6) tissue 0.1(0.2) 0.1 (0.2) scavenger filter 0.4 (0.4) 0.4 (0.4) AeroDose-specific:box 1.0 (0.9) 1.0 (0.9) tissue 0.0 (0.1) 0.0 (0.1) *Wilcoxonmatched-pairs signed ranks test on intent to treat dataset. ExcludingSubject 1007: p = 0.04. Statistical significance: p ≦ 0.05.Nebulization Time

The nebulization time (i.e., time required from first tidal breath untilthe nebulizer ran dry) was significantly shorter (p=0.005) for theAerodose delivery system (mean±SD=5.70±1.16 minutes) than for the PARILC PLUS system (20.40±3.47 minutes) (Table 14 below).

TABLE 14 MEAN (SD) NEBULIZATION TIME Intent to Treat (n = 10)Nebulization Time* TOBI 300 mg TOBI 60 mg Parameter PARI LC PLUSAeroDose p-value Nebulization Time (minutes): Mean 20.40 5.70 0.005 SD3.47 1.16 Minimum 17.0 4.0 Maximum 29.0 8.0 no. subjects 10 10Serum Tobramycin Concentrations and Pharmacokinetic Parameters

Administration of TOBI 300 mg using the PARI LC PLUS delivery systemproduced higher mean serum tobramycin concentrations, a higher meanC_(max), and a greater AUC₍₀₋₈₎ than administration of TOBI 60 mg usingthe Aerodose delivery system. The mean time to maximum tobramycinconcentration (T_(max)) was similar for the two delivery systems.

Serum tobramycin concentrations for all subjects were below quantifiablelimits before dosing in both period 1 and period 2.

After dosing, two subjects had serum tobramycin concentrations thatcould not be measured (i.e., results were below the quantifiable limitof 0.20 μg/mL) during one of the two treatment periods. These twosubjects were inevaluable for pharmacokinetic analysis during the periodindicated but provided evaluable results for the alternate period.

Consistent with the high efficiency of the Aerodose system, mean serumtobramycin concentrations were slightly lower throughout the 8-hourpostdose observation period after delivery of TOBI 60 mg using theAerodose system than after delivery of TOBI 300 mg using the PARI LCPLUS system (Table 15 below). Maximum plasma concentrations for bothregimens were reached within 2 hours after the end of inhalation (TOBI300 mg and PARI inhaler: 1 hr and 2 hr means=0.63 μg/mL; TOBI 60 mg andAerodose inhaler: 2 hr mean=0.48 μg/mL). By 8 hours after the end ofinhalation, the plasma concentrations were below the limit ofquantitation in 5 subjects after the Aerodose inhaler and in twosubjects after the PARI LC PLUS nebulizer.

TABLE 15 SERUM TOBRAMYCIN CONCENTRATIONS AND PHARMACOKINETIC PARAMETERSIntent to Treat (n = 10) TOBI 300 mg TOBI 60 mg Parameter* PARI LCPLUS^(a) AeroDose^(b) Serum Tobramycin (μg/mL): Time Before and AfterDosing: Predose  0.00 (0.00) 9  0.00 (0.00) 9 30 minutes  0.42 (0.24) 9 0.22 (0.23) 9 1 hour  0.63 (0.29) 9  0.41 (0.22) 9 2 hours  0.63 (0.25)9  0.48 (0.20) 9 4 hours  0.50 (0.16) 9  0.38 (0.10) 9 8 hours  0.22(0.14) 9  0.13 (0.12) 9 Pharmacokinetic Parameters: C_(max) (μg/mL)0.677 (0.279) 9 0.482 (0.201) 9 T_(max) (hr) 2.213 (0.923) 9 2.207(0.788) 9 T_(1/2) (hr) 4.269 (1.058) 9 6.071 (3.357) 9 AUC₍₀₋₈₎ (μg/mL ·hr) 3.622 (1.319) 9 2.553 (0.989) 9 AUC_((0-∞)) (μg/mL · hr) 5.273(1.699) 9 4.630 (0.967) 9 Pharmacokinetic Parameters Normalized to Dose:C_(max) (μg/mL)/mg 0.002 (0.001) 9 0.008 (0.003) 9 AUC₍₀₋₈₎ (μg/mL ·hr)/mg 0.012 (0.004) 9 0.043 (0.016) 9 AUC_((0-∞)) (μg/mL · hr)/mg 0.018(0.006) 9 0.077 (0.016) 9 *Cell entries are mean, (SD), no. of subjects.^(a)TOBI 300 mg summary statistics exclude BQL results for Subject 1007throughout period 2. ^(b)TOBI 60 mg summary statistics exclude BQLresults for Subject 1006 throughout period 1.Pharmacokinetic Results

The mean of the maximum tobramycin concentrations for all subjects(C_(max) in Table 15 above) was greater after TOBI 300 mg delivered bythe PARI LC PLUS system (mean±SD=0.677±0.279 μg/mL) than after TOBI 60mg delivered by the Aerodose system (0.482±0.201 μg/mL). This meandifference in log C_(max) was statistically significant (p=0.0018), andthere was no evidence to suggest the presence of a carryover effect inC_(max) (p=0.6400). The Aerodose inhaler was more efficient than thePARI LC PLUS nebulizer based on C_(max) results adjusted for TOBI doseadministered (TOBI 300 mg with PARI LC PLUS=0.002±0.001 (μg/mL)/mg; TOBI60 mg with Aerodose=0.008±0.003 (μg/mL)/mg).

The time to maximum tobramycin concentrations (T_(max)) was virtuallyidentical for the two delivery systems (mean=2.213 hours for PARI LCPLUS and 2.207 hours for Aerodose systems in Table 15 above). T_(max)results in the present study were consistent with observations in aprevious study¹⁵ that peak serum tobramycin concentrations occurred at 1to 2 hours after inhalation.

The mean elimination half-life (T_(1/2)) was 4.269 hours for the PARI LCPLUS system and 6.071 hours for the Aerodose system (Table 7).

The mean area under the serum concentration-time curve through 8 hourspostdose (AUC₍₀₋₈₎) was significantly greater (p=0.0002 on log AUC₍₀₋₈₎)after TOBI 300 mg delivered by the PARI LC PLUS system (3.622±1.319μg/mL•hr) than after TOBI 60 mg delivered by the Aerodose system(2.553±0.989 μg/mL•hr). There was no evidence (p=0.7858) to suggest thepresence of carryover effect in AUC₍₀₋₈₎. The greater efficiency of theAerodose inhaler was also seen in dose-normalized AUC₍₀₋₈₎ results (TOBI300 mg with PARI LC PLUS=0.012±0.004 [μg/mL•hr]/mg; TOBI 60 mg withAerodose=0.043±0.16 [μg/mL•hr]/mg).

The mean area under the serum concentration by time curve extrapolatedto infinity (AUC_((0-∞)) in Table 7 above) was not significantlydifferent (p=0.5477 on log AUC_((0-∞))) after administration of TOBI 300mg using the PARI system (5.273±1.699 μg/mL•hr) than afteradministration of TOBI 60 mg using the Aerodose system (4.630±0.967μg/mL•hr). No carryover effect was detected (p=0.6006). The greaterefficiency of the Aerodose inhaler was similarly seen in dose-normalizedAUC_((0-∞)) results (TOBI 300 mg with PARI LC PLUS=0.018±0.006[μg/mL•hr]/mg; TOBI 60 mg with Aerodose=0.077±0.16 [μg/mL•hr]/mg).

Unplanned, exploratory analyses suggested that female subjects achievedslightly higher C_(max), AUC₍₀₋₈₎ and AUC_((0-∞)) results than malesubjects after both TOBI 300 mg and TOBI 60 mg treatments.

Extent of Exposure

The duration of exposure to study drug and the dose of study drug werenot varied in this study. All 10 subjects received a single 300 mg (5mL) TOBI dose using the PARI LC PLUS jet nebulizer with the DeVilbissPulmoAide compressor delivery system (control treatment) on one occasionand a single 60 mg (1 mL) TOBI dose using the Aerodose inhaler(experimental treatment) on a second occasion. Each dose wasradiolabeled with up to 10MBq ^(99m)Tc-DTPA and administered in arandomized two-way crossover fashion separated by a 44-hour minimumwashout period.

The mean whole lung deposition using the PARI LC PLUS nebulizer was 8.2%(24.5 mg) of the 300 mg TOBI dose. The mean whole lung deposition usingthe Aerodose inhaler was 34.8% (20.9 mg) of the 60 mg TOBI dose. A meanof 14.4% (43.3 mg) and 31.5% (18.9 mg) of the corresponding doses weredeposited in the oropharynx using the PARI LC PLUS and Aerodoseinhalers, respectively. Both inhaler systems were configured such thateach subject's exhaled material was collected and did not escape withradioactive aerosol into the surrounding atmosphere. The PARI LC PLUSnebulizer also included a system to collect any radiolabeled dropletsescaping from the nebulizer.

Bronchospasm

In this study, decreases in the relative FEV₁% predicted ≧10% (notclinically significant if <20%) and ≦20% (clinically significant) frompredose measurements to 30-minutes postdose measurements with eachdelivery system were used as indicators of bronchospasm (airwayreactivity). Reductions in FEV₁% predicted ≧20% were consideredclinically significant for the purposes of the study. No subject had adrop in FEV₁% predicted ≧10% from predose to postdose regardless ofdelivery system during this study.

Discussion and Overall Conclusions

The study of this example demonstrates that the AeroDose™ inhaler wasmore efficient in delivery of aerosolized tobramycin to the lungs ofhealthy adult volunteers than the approved PARI LC PLUS jet nebulizerwith DeVilbiss PulmoAide compressor. Since the Aerodose inhaler isbreath-actuated and generates aerosol only during inhalation,proportionally more of the Aerodose dose should be delivered to thelungs than is delivered by the PARI LC PLUS, and there should be minimalwastage of drug by aerosolization during exhalation or by incompleteaerosolization of the contents of the drug reservoir.

During the study, the Aerodose inhaler delivered a significantly greaterpercentage of the dose to the lungs than the PARI LC PLUS nebulizer(mean 34.8% vs. 8.2%: p=0.005). The actual amount of the dose depositedin the lungs was slightly but not significantly less using the Aerodoseinhaler than using the PARI LC PLUS nebulizer (20.9 mg vs. 24.5 mg:p=0.202). These data demonstrate that the Aerodose inhaler deliverednearly as much tobramycin to the lungs as the PARI LC PLUS nebulizerdespite nebulizing one-fifth the amount of tobramycin.

Approximately 32% of the Aerodose dose was wasted on the inhaler andexhalation filter combined. By contrast, more than 74% of the PARI LCPLUS dose was wasted by deposition on the inhaler and exhalation filter.

When the Aerodose inhaler was used, 15.2% (9.1 mg) of the 60 mg TOBIdose remained deposited on the inhaler, and 16.9% (10.1 mg) wasdeposited on the exhalation filter. Since no aerosolization occurredduring exhalation when the Aerodose was used, the observed depositioncould have been due only to seepage through the mouth-inhaler seal or toresidual radiolabeled tobramycin inhaled but immediately exhaled and notdeposited in either the lungs or the oropharynx (including esophagus andstomach). Four subjects were noted to have either experienced problemsmaintaining a seal around the mouthpiece of the Aerodose inhaler orreported that the inhaler failed to nebulize one of the two aliquots ofthe dose solution. These subjects had approximately 47%, 19%, 53%, and26%, respectively, of the 60 mg dose deposited on the inhaler andexhalation filter combined. The highest two of these figures were abovethe range noted for the rest of the subjects (ranging from 17% to 40%deposited on inhaler and exhalation filter combined). Problems withincomplete nebulization or wide variation in subject inhalationeffectiveness may have contributed to the amount of wastage of drugduring Aerodose usage in the present study.

By comparison, when the PARI LC PLUS jet nebulizer was used, 42.6%(127.8 mg) of the 300 mg TOBI dose remained deposited on the inhaler,and 31.6% (94.8 mg) was deposited on the exhalation filter. Presumably,most or all of the exhalation filter deposition was due to continuedaerosolization and consequent loss of drug while subjects exhaled.

Thus, both the Aerodose inhalers and PARI LC PLUS nebulizers wasted drugproduct in the present study by reason of retention of radiolabeled drugon or in the inhaler or deposition of drug on the exhalation filter (anaverage of approximately 19 of 60 mg wasted when the Aerodose inhalerwas used and approximately 223 of 300 mg wasted when the PARI LC PLUSnebulizer was used). The proportion of the total dose wasted using theAerodose inhaler was less than half of that wasted using the approvedPARI LC PLUS nebulizer.

The Aerodose inhaler also appeared to exhibit better “targeting” ordelivery of the dose to the lungs, the target site of the usual P.aeruginosa infection in cystic fibrosis patients, than the PARI LC PLUSnebulizer. The Aerodose inhaler deposited slightly more tobramycin inthe lungs than in the oropharynx, esophagus, and stomach (lungs 34.8%vs. 31.5% of the 60 mg dose). By comparison, the PARI LC PLUS nebulizerdeposited proportionally less of the dose in the lungs than inoropharynx, esophagus, and stomach (lungs 8.2% vs. 14.4% of the 300 mgdose). The ratio of lung to oropharyngeal, esophagus, and stomachcombined was approximately 1.1 for the Aerodose inhaler and 0.6 for thePARI LC PLUS nebulizer.

In addition to greater efficiency by greater delivery of drug to thelungs and proportionally greater targeting of the lungs, the Aerodoseinhaler was also anticipated to be more efficient by reason ofproportionally greater delivery of tobramycin to peripheral rather thancentral lung regions. The Aerodose particle MMD is smaller (mean MMD=4.0μm) than that produced by the PARI LC PLUS nebulizer (mean MMD=4.8 μm),so the expectation was that the Aerodose inhaler would deposit a greaterproportion of aerosol generated during inhalation in the peripheralairways than the PARI LC PLUS. During the study, the Aerodose inhalerdeposited 13.2% (7.9 mg) of the 60 mg dose in the peripheral airways,while the PARI LC PLUS nebulizer deposited 3.1% (9.3 mg) in peripheralairways. Although the Aerodose inhaler achieved proportionally greaterperipheral deposition than the PARI LC PLUS nebulizer, both inhalersfell short of amounts predicted for peripheral deposition based ontheoretical considerations (Aerodose estimated to peripherally deposit60% (36 mg) of the 60 mg dose=1.0 mL fill volume•0.95aerosolization•0.62 respirable particles; PARI LC PLUS estimated toperipherally deposit 16% (48 mg) of the 300 mg dose=5.0 mL fillvolume•0.64 aerosolization•0.44 respirable particles).

Results of the study also showed that the Aerodose inhaler requiredsignificantly less nebulization time than the PARI LC PLUS nebulizer(mean 20.4 vs. 5.7 minutes, respectively). The 5.7 minute averagenebulization time for the Aerodose inhaler did not include the amount oftime needed to fill the drug reservoir before nebulization of the secondaliquot. Based on nebulization time results and other inhaler featuresincluding portability, ease of use, and lack of a need for a compressor,it is anticipated that the Aerodose inhaler would improve patientcompliance.

Serum tobramycin concentrations, maximum concentrations, and extent ofabsorption were greater after administration of TOBI 300 mg using thePARI LC PLUS nebulizer than after administration of TOBI 60 mg using theAerodose inhaler. These results appeared to be consistent with amountsof tobramycin deposited in lungs and oropharynx (including esophagus andstomach) combined where systemic absorption occurred (mean tobramycindeposited in lungs and oropharynx combined=67.8 mg after TOBI 300 mg;mean=39.8 mg after TOBI 60 mg). Mean serum tobramycin concentrationswere higher throughout the 8-hour observation period afteradministration of TOBI 300 mg using the PARI LC PLUS nebulizer thanafter administration of TOBI 60 mg using the Aerodose inhaler. MeanC_(max) values were 0.677 and 0.482 μg/mL for TOBI 300 mg and TOBI 60mg, respectively (statistically significant: p=0.0018). Mean T_(max)results for both inhalers were virtually identical (2.213 and 2.207hours, respectively). Apparent absorption of tobramycin wassignificantly greater during the 8-hour postdose period after TOBI 300mg than after TOBI 60 mg (mean-AUC₀₋₈=3.622 and 2.553 μg/mL•hr,respectively; statistically significant: p=0.0002), but no treatmentdifferences were noted in AUC_(0-∞) (TOBI 300 mg and TOBI 60 mgmeans=5.273 and 4.630 μg/mL•hr, respectively; p=0.5499).

Current results suggested that the 60 mg TOBI dose aerosolized using theAerodose inhaler produced tobramycin deposition and serum tobramycinconcentration results that were significantly or substantially less thanresults obtained after aerosolization of the approved TOBI 300 mg doseusing the PARI LC PLUS nebulizer. Normalized for administered dose, theAerodose inhaler was substantially more efficient on a per milligrambasis in delivery of tobramycin to the systemic circulation than thePARI LC PLUS nebulizer. These results are consistent with the higherdeposition (on a milligram basis) in the lung.

Results of the study also showed that single doses of TOBI 300 mgdelivered using the PARI LC PLUS jet nebulizer and of TOBI 60 mgdelivered using the Aerodose breath actuated nebulizer were safe andwell-tolerated by healthy adult male and female volunteers. No instancesof bronchospasm were observed, and no notable quantitative changes inpulmonary function were seen. No notable adverse events (AEs) werereported by subjects, and there were no apparent differences betweentreatment groups in incidence of any AE. Six treatment emergent AEs werereported by 4 subjects, but all events were mild in intensity. Twoinstances of headache were considered possibly or definitely related totreatment. No clinically significant laboratory results or changes inresults were observed. No adverse vital signs, body weights, physicalfindings, or electrocardiogram results were observed. No evidence ofsystemic toxicity, as measured by unusually high serum tobramycinconcentrations, was observed.

Example 3 In Vivo Study 2

A comparison was made of the safety, pharmacokinetics, aerosol deliverycharacteristics, and nebulization time of the conventional dose andinhalation delivery system (5 mL ampoule containing 300 mg tobramycinand 11.25 mg sodium chloride in sterile water for injection (TOBI®tobramycin solution for inhalation, Chiron Corporation, Seattle, Wash.),pH 6.0; administered with a PARI LC PLUS™ jet nebulizer with a DeVilbissPulmoAide™ compressor set to deliver an output pressure of 20 psi—the“control delivery system”) with a dose of 420 mg Tobramycin Solution forInhalation at 120 mg/mL (excipient 3.5 mL of ¼ normal saline adjusted toa pH of 6.0±0.5; 420 mg in 3.5 mL) delivered by the PARI LC PLUS™ jetnebulizer with a Invacare MOBILIARE™ compressor set to deliver an outputpressure of 35 psi (the “experimental delivery system”).

The study was designed as an open label, randomized, single-dose,multicenter, two treatment, active-control, and parallel trial. Eachpatient was administered a single aerosolized dose of study drug witheither the control delivery system or the experimental delivery system.In accordance with the study design, a total of 36 eligible male andfemale patients 12 years of age or older with a confirmed diagnosis ofcystic fibrosis were enrolled with a minimum of 4 patients at each site.A 2:1 randomization ratio was employed for assignment of patients to thetreatment groups. In the presence of the investigator or studycoordinator, each patient was to self-administer either a single dose of300 mg TOBI® with the control delivery treatment or a single dose of 420mg Tobramycin Solution for Inhalation with the experimental deliverytreatment as listed below.

Control Treatment:

Aerosolized 300 mg TOBI® was delivered by PARI LC PLUS jetnebulizer/DeVilbiss PulmoAide compressor: Preservative free tobramycinfor inhalation 60 mg/mL (excipient 5 mL of ¼ normal saline adjusted to apH of 6.0±0.5); 300 mg in 5 mL; lot number 03K1C (TOBI® at 60 mg/mL).

Experimental Treatment (420 mg Tobramycin Solution for Inhalation or“TSI”):

Aerosolized 420 mg Tobramycin Solution for Inhalation (TSI) wasdelivered by PARI LC PLUS jet nebulizer/Invacare MOBILAIRE compressor:Preservative free tobramycin 120 mg/mL (excipient 3.5 mL of ¼ normalsaline adjusted to a pH of 6.0±0.5); 420 mg in 3.5 mL.

Both 300 mg TOBI® and 420 mg Tobramycin Solution for Inhalation aresterile, non-pyrogenic, preservative-free antibiotics prepared foraerosolization. Each mL of TOBI® contains 60 mg tobramycin and 2.25 mgsodium chloride in sterile water for injection, pH 6.0±0.5 (controltreatment). Each mL of TSI contains 120 mg tobramycin and 2.25 mg sodiumchloride in sterile water for injection, pH 6.0±0.5 (experimentaltreatment). Drug supplies for this study were manufactured by AutomatedLiquid Packaging (ALP), Woodstock, Ill. All repackaging, labeling, anddistribution for clinical use was provided by Packaging Coordinators,Inc. (PCI), Philadelphia, Pa. Study drug and device supplies wereshipped from Chiron Corporation, Emeryville, Calif. for each patientupon enrollment in the study.

The duration of study participation for each patient was approximatelytwo weeks including a brief (one day one week before treatment)screening period, one day treatment period, and a follow-up one-weekafter treatment. Study treatments were evaluated for safety and aerosoldelivery characteristics up to eight hours post-dose on the day of thesingle dose treatment administration. The patient was to return to theclinic for a seven day post-treatment follow-up assessment of safety.There were no planned interim safety analyses.

Criteria for Evaluation:

Safety:

-   -   Incidence of bronchospasm defined as FEV₁ decrease of ≧10% and        FEV₁ decrease of ≧20% from predose to 30 minutes postdose;    -   Relative change and absolute change in airway response (FEV₁)        after single dose of study drug;    -   Laboratory measures of safety (clinical lab tests, spirometry        testing);    -   Incidence of treatment emergent adverse events.        Aerosol Delivery:    -   Pharmacokinetic assessment of sputum and serum tobramycin        concentrations;        -   Sputum was collected at pre-dose and 15 minutes, 1, 2, 4,            and 8 hours after dosing;        -   Serum was collected at pre-dose and 10 minutes, 1, 2, 4, 6,            and 8 hours after dosing;    -   Nebulization time.

Statistical methods: All patients who received a dose of study treatmentwere evaluated for safety and aerosol delivery characteristics.

Rate of bronchospasm measured by the percent of patients with ≧10% and≧20% relative decrease in FEV₁% from pre-dose to 30 minutes post-dosewas summarized and compared between treatments using the Fisher's exacttest.

A two sample t-test was used to compare the relative change in FEV₁%from predose to 30 minutes postdose between experimental and controltreatments. Summary statistics for relative and absolute change in FEV₁were tabulated by treatment.

Sputum and serum area under curve (AUC₀₋₈) and maximum concentrations(C_(max)) were summarized and analyzed for treatment differences using ageneral linear model analysis of variance (ANOVA). Pharmacokineticparameters were calculated using a noncompartmental model. Sputum andserum concentrations were summarized and graphically illustrated bytreatment.

Laboratory measures of safety and incidence of treatment-emergentadverse events were summarized and descriptively compared betweentreatments.

Nebulization time was recorded and summarized for each of the twodelivery treatments.

Safety Variables

Aerosol delivery variables were tobramycin concentrations in sputum andserum, sputum and serum tobramycin pharmacokinetic parameters, andaerosol nebulization time. Safety variables were the incidence andseverity of bronchospasm, measured as the number of patientsexperiencing a ≧10% and a ≧20% decrease in forced expiratory volume inone second (FEV₁) within 30 minutes after dosing (a ≧20% decrease inFEV₁ was considered clinically significant), the incidence of treatmentemergent adverse events (AEs), clinical laboratory test results, thenumber of patients with serum tobramycin concentrations ≧4 μg/mL,physical examination findings, and vital signs results.

Primary Aerosol Delivery Variables

Evaluation of the aerosol delivery characteristics of 420 mg TobramycinSolution for Inhalation at 120 mg/mL delivered by the PARI LCPLUS™/Invacare MOBILAIRE™ delivery system compared to 300 mg TOBI® at 60mg/mL delivered by the FDA-approved PARI LC PLUS™/DeVilbiss PulmoAide™delivery system was based on determination of sputum and serumtobramycin concentrations, calculation of certain sputum and serumpharmacokinetic parameters, and measurement of nebulization time.

Sputum Tobramycin Concentrations: Sputum samples were expectorated bypatients from a deep cough and collected before day 1 dosing (predose)and at 0.25, 1, 2, 4, and 8 h after the end of the nebulization period.Sputum samples were collected as close as possible to specified timesand were considered to have been drawn on time within ±2 minutes for the15-minute posttreatment collection and within ±10 minutes for the 1-,2-, 4-, and 8-hour posttreatment collections. Samples collected outsidethese intervals were considered protocol deviations. A minimum 100 mgsputum (not saliva) sample was collected before the single dose of studytreatment to determine the baseline tobramycin concentration.Immediately after dosing, patients rinsed their mouths with 30 mL ofnormal saline, gargled for 5-10 seconds, and expectorated the rinse.This sequence of post-treatment rinsing was repeated for a total ofthree rinses. Sputum samples were stored at −70° C. or below untilanalysis. The concentration of tobramycin was analyzed usingreversed-phase high-performance liquid chromatography (HPLC) withultraviolet detection. Patient sputum samples were first liquefied with0.2 N NaOH and diluted with Tris buffer (20.0 g Trizma base/L). Sputumstandard samples were prepared by spiking diluted pooled sputum from CFpatients with tobramycin to final concentrations of 0, 20, 40, 100, 200,400, and 1000 μg/g of sputum. Assay quality control samples wereprepared by spiking diluted pooled sputum to contain 40, 300, and 800μg/g. The internal standard sisomycin (100 μL, 0.15 mg/mL in Trisbuffer) was added to 100 μL of each standard, control, and subjectsample, followed by 400 μL of acetonitrile and 50 μL of2,4-dinitrofluorobenzene (0.17 g/mL). The sample reaction mixtures wereheated in a dry-block heater for 1 h at 80° C. After addition of 600 μLof 60/40 acetonitrile/water (v/v), 50 μL was analyzed by HPLC. Sampleswere injected onto a Waters Nova-Pak® C-18, 3.9×150 mm, 4 μm columnconnected to a Waters HPLC with 600E pump, 486 or 2487 ultravioletdetector (λ_(max)=360 nm) and 717 Plus autosampler. The mobile phaseconsisted of 0.2% acetic acid in acetonitrile (39/61, v/v), pumped at arate of 1.5 mL/min for 5 min, 2.0 mL/min for an additional 9 or 10 min,depending on the length of the run. Waters Millennium-32 C/S LC Software(version 3.20) was used to operate the Waters HPLC instruments as wellas acquire raw data, process, compute, and report the analyticalresults. The ratio of the peak height of tobramycin to the internalstandard sisomycin (PHR) was calculated. The assay was completed in 8runs. Retention time ranges of 4.2 to 4.4 min, and 10.8 to 11.8 min wereobserved for tobramycin and sisomycin, respectively. A linearrelationship existed between PHR and concentration from 20 to 1000 μg/gfor sputum. The regression model was PHR=Bx+A (x=tobramycinconcentration), weighted 1/x. The lower limit of quantitation was 20μg/g. The concentrations of the standard samples were within 97 to 105%of the nominal concentration, with coefficients of variation not higherthan 3.4%. The precision of the assay, as reflected by the CV of thequality control samples, was 2.3%, 2.2% and 2.6%, for the 40, 300, and800 μg/g samples, respectively. The accuracy of the method, reflected bythe interassay recoveries of the quality control samples, was 103%, 99%,and 98% for the 40, 300, and 800 μg/g quality control samples,respectively. Overall, this method exhibited suitable accuracy andprecision for pharmacokinetic analysis.

Serum Tobramycin Concentrations: Blood samples were collected at predoseand at 0.167, 1, 2, 4, 6, and 8 h after the end of the nebulizationperiod. Samples were collected as close as possible to specified timesand were considered to have been drawn on time within ±2 minutes for the10-minute posttreatment collection and within ±10 minutes for the 1-,2-, 4-, 6-, and 8-hour posttreatment collections. Samples collectedoutside these intervals were considered protocol deviations. Serum washarvested and stored at −70° C. or below until analysis. Concentrationsof tobramycin in serum were analyzed with a modified fluorescencepolarization immunoassay (FPIA) method using the Abbott TDx®/TDxFLx®System. Samples were added directly to the dilution well of the samplecartridge. The net polarization was acquired by the TDx®/TDxFLx®apparatus and manually entered into an Oracle database. A weighted fourparameter logistic equation was used to calculate the concentrations oftobramycin. The concentrations of tobramycin were reported in terms offree base equivalents. For assaying the subject samples of the study,calibration standards (0.050, 0.100, 0.200, 0.400, 0.600, 0.800, 1.000μg/mL) and quality control samples (0.150, 0.400, and 0.750 μg/mL) wereprepared in house. The assay was completed in 8 runs. A linearrelationship existed between polarization response and concentrationfrom 0.050 μg/mL to 1.00 μg/mL. The lower limit of quantitation was0.050 μg/mL. The precision of the assay, as reflected by the CV of thequality control samples, was 3.3%, 4.9%, and 4.9% for the 0.150, 0.400,and 0.750 μg/mL samples, respectively. The accuracy of the method,reflected by the mean interassay recoveries of the quality controlsamples, was 101%, 103%, and 104% for the 0.150, 0.400, and 0.750 μg/mLsamples, respectively. Overall, this method exhibited suitable accuracyand precision for pharmacokinetic analysis.

Nebulization Time:

Nebulization time was defined as the length of time from the start ofthe patient's first tidal breath to completion of aerosoladministration. Aerosol administration was complete when the nebulizerbegan to sputter. If aerosol administration was interrupted for anyreason, the time of interruption and start and stop times of continuedaerosol administration were recorded. If dosing was interrupted,nebulization time was considered to be not calculable.

Residual Tobramycin in the Nebulizer: The amount of residual tobramycinsolution remaining in the nebulizer after completion of aerosoladministration was determined by recording pretreatment andposttreatment weight of the nebulizer system including nebulizer, filtervalve, and study drug. The research coordinator collected residual studydrug remaining in the nebulizer after aerosol administration into a viallabeled with patient information. The vial was returned for measurementof the amount of drug output from the nebulizer and for determination ofthe extent of the concentration of study drug left in the nebulizer.

Safety Variables

Bronchospasm: The study protocol prospectively identified bronchospasmas an adverse airway response to inhalation of aerosolized antibiotic ofparticular relevance to patients with cystic fibrosis. In order todetermine whether current study treatments produced bronchospasm,patients performed spirometry (pulmonary function) tests to measure FEV₁before and 30 minutes following completion of study treatmentadministration according to the method described in the protocol. Airwayresponse to the study drug was assessed by evaluating the relativepercent change in FEV₁ from predose to 30 minutes after the end oftreatment using the following formula.${{relative}\quad{FEV}_{1}\quad\%\quad{change}} = {\frac{{30\quad\min\quad{postdose}\quad{FEV}_{1}} - {{predose}\quad{FEV}_{1}}}{{predose}\quad{FEV}_{1}} \times 100\quad\%}$

Bronchospasm was defined as a decrease in FEV₁ of ≧10% at 30 minutesafter dosing, relative to the predose result. A decrease in FEV₁ of ≧20%was considered to represent clinically significant bronchospasm.Moreover, if there was a posttreatment decrease in FEV₁ of ≧30%,spirometry was to be repeated until the FEV₁ decrease was <10% below thepredose result. An FEV₁% decrease ≧30%, and all symptoms associated withthe change in pulmonary function, were to be recorded as adverse events.The protocol defined the severity of decrease in FEV₁ based in part onthe National Cancer Institute (NCI) Common Toxicity Criteria AdverseEvents Grading Scale. However, slight inconsistencies in the protocoldefinitions of bronchospasm and of the severity of FEV₁ changes werenoted during preparation of the analyses and report. To resolve thedifferences, the actual system used during the analysis to classify theseverity of FEV₁ changes relative to the predose result is listed below.

TABLE 16 AIRWAY RESPONSE (FEV₁) (BRONCHOSPASM) FEV₁ % DECREASE BELOWPREDOSE VALUE Severity Protocol Classification Analysis ClassificationMild: ≧10%-≦20% ≧10%-<20% Moderate: >20%-≦30% ≧20%-<30% Severe: >30%≧30%Clinical Laboratory Tests

At screening, laboratory tests were performed to measure serumcreatinine, blood urea nitrogen (BUN), urine protein (proteinuria bydipstick), and to detect pregnancy in females of childbearing potential.If abnormal at screening, serum creatinine, BUN, and urine protein testswere to be repeated before the time of dosing. Final test results wereobtained based on specimens drawn at the follow-up visit on day 8 of thestudy.

After the mean body weight difference between treatment groups becameknown by Chiron personnel, estimated creatinine clearance was calculatedfor patients using the Cockroft-Gault equation below to evaluate renalclearance characteristics of the two groups and to clarify thepharmacokinetic results of the study.

Male Patients:

estimated creatinine

clearance (mL/min)=(140-age[yr])(body weight[kg])/72*(serum creatinine[mg/dL])

Female Patients:

estimated creatinine

clearance (mL/min)=0.85*((140-age[yr])(body weight[kg])/72*(serumcreatinine [mg/dL])

All abnormal laboratory test results, whether present on entry into thestudy or arising during the study, were evaluated by the studyinvestigator for clinical significance and relationship to study drug.If the abnormal result was considered unrelated to study drug, theinvestigator was to identify the probable cause of the result.Laboratory results considered markedly abnormal and clinicallysignificant were BUN>16 mmole/l (>45 mg/dl), serum creatinine>177μmole/l (>2 mg/dl), and proteinurea≧3+.

Other Safety Variables

Serum assay results were screened for tobramycin concentrations ≧4 μg/mLfrom specimens collected from 10 minutes through 8 hours aftercompletion of study treatments. In parallel, patient records and CRFswere examined for evidence of systemic toxicity potentially related toelevated tobramycin levels. Assay results were not available until afterpatients' discharge from the study, so screening for unusually highserum tobramycin concentrations and evidence of systemic toxicity wasundertaken when all pertinent results were received.

Pharmacokinetics

Pharmacokinetic parameters for both sputum and serum tobramycin werederived to characterize aerosol delivery capabilities of control andexperimental treatments. The concentration (C) versus time (t) data(Listings 16.2.5.2 and 16.2.5.3) were analyzed by model-independentmethods to obtain the pharmacokinetic parameters. The areas under theplasma concentration-time curve from time zero (predose) to infinity(AUC) and under the first moment of the plasma concentration-time curve(AUMC) were obtained by the trapezoidal rule, extrapolated to infinity.The terminal rate constant (λ_(z)) was determined by log-linearregression of the terminal phase. The maximum concentration (C_(max))and the time to maximum after the end of the nebulization period(t_(max)) were obtained by inspection. In addition, the followingparameters were calculated:t _(1/2)=ln(2)/λ_(z)CL/F=D/AUCV _(z) /F=CL _(po)/λ_(z)where t_(1/2) is the terminal half-life, CL/F is the total bodyclearance, and Vz/F is the terminal volume of distribution. Since theabsolute bioavailability of tobramycin (F) in the two formulations usedin this study is not known, the calculated clearance and volume ofdistribution are hybrid parameters that do not account for differencesin bioavailability between the two formulations. All parameters werecalculated for serum; only AUC, C_(max), t_(max), λ_(z), and t_(1/2)were calculated for sputum.

Concentrations below the lower limit of quantitation were treated aszero for all calculations. Since there was an insufficient volume ofmatrix to assay tobramycin in the following time points, they wereexcluded from the pharmacokinetic analysis:

TABLE 17 EXCLUSIONS FROM PHARMACOKINETIC ANALYSIS Matrix Subject TimeSerum 01-110 6 02-116 1 03-102 0.167, 1, 2 03-105 0, 0.167, 1, 2, 4, 6,8 03-131 0.167 05-125 4, 8 06-120 2 Sputum 08-127 2Data Handling

Case report form data were entered in duplicate into a Clintrial™database by the department of Biostatistics and Clinical Data Management(BCDM) at Chiron Corporation. Data quality assurance was performed usingPL/SQL and SAS™ 6.12 or higher software (SAS Institute, Cary, N.C.).Analysis was performed by Chiron Corporation, using SAS version 6.12 orhigher software, based on a predefined analysis plan developed by ChironCorporation. The estimated overall database error rate was 0.xx% with anupper 95% confidence limit of 0.xx%. This upper confidence limit isbelow the departmental standard of 0.5%.

Statistical Methods and Determination of Sample Size

Statistical and Analytical Plans: Serum and sputum pharmacokineticparameters, the incidence of bronchospasm, and the relative change frompredose in 30-minute postdose FEV₁% predicted were analyzedstatistically to assess the significance of any apparent differencesbetween test and reference treatments. All statistical tests describedin following sections were two-tailed tests of significance, and thecriterion for statistical significance was set at α=0.05 unlessotherwise noted.

Primary Aerosol Delivery Analyses:

All patients who received the single dose of test or reference treatmentwere included in the analysis and evaluation of aerosol deliverycharacteristics. Aerosol delivery was characterized on the basis ofserum and sputum tobramycin concentrations, derived serum and sputumpharmacokinetic parameters, and nebulization time. The effect oftreatment (300 mg TOBI vs 420 mg TSI), gender, and age group (less than18, 18 years or older) on the AUC, C_(max), λ_(z), CL/F, and Vz/F oftobramycin in serum, and on the AUC, C_(max), and λ_(z) of tobramycin insputum, was analyzed by a three-way analysis of variance. Furthermore,the relationship between body weight and AUC, C_(max), CL/F, and Vz/F oftobramycin in serum, and between body weight and AUC and C_(max) oftobramycin in sputum were analyzed by regression analysis. All testsemployed a significance level α=0.05. All parameters are expressed asthe mean±SD. A harmonic half-life was estimated as:{overscore (t _(1/2) )}=ln(2)/{overscore (λ_(z))}

-   -   in which {overscore (λ_(z))} is the arithmetic mean of the        terminal rate constants at each dose. The standard deviation of        the harmonic half-life, SD({overscore (t_(1/2))}), was obtained        as:        ${{SD}\left( \overset{\_}{t_{1/2}} \right)} = {\frac{\ln(2)}{\overset{\_}{\lambda_{z}}} \times \frac{{SD}\left( \overset{\_}{\lambda_{z}} \right)}{\overset{\_}{\lambda_{z}}}}$        where SD({overscore (λ_(z))}) is the standard error of the mean        terminal rate constant at each dose.        Safety Analyses

Analysis of Airway Response:

The primary safety variable was the rate of bronchospasm, defined as a≧10% decrease in FEV₁ from predose to 30 minutes after treatment on day1 of the study. Secondary safety variables were (a) the rate ofclinically significant bronchospasm, defined as a ≧20% decrease in FEV₁from predose to 30 minutes after treatment on day 1, and (b) therelative change in FEV₁ from predose to 30 minutes after treatment onday 1. The rates of occurrence of all instances of bronchospasm (FEV₁%decrease ≧10%) and of all instances of clinically significantbronchospasm (FEV₁% decrease ≧20%) were analyzed to assess thestatistical significance of test vs. reference treatment differencesusing the Fisher's Exact test. The protocol specified that the treatmentdifference in the incidence of bronchospasm would be tested forstatistical significance using the Cochran-Mantel-Haenszel test. Due tothe low incidence of bronchospasm in the enrolled patients, the Fisher'sexact test was used for this analysis since it makes no assumptionsregarding the minimum expected cell frequencies. The test vs. referencetreatment difference in mean relative change from predose in 30-minutepostdose FEV₁% predicted was tested for statistical significance usingthe two-sample t-test.

Adverse Events: The total incidence of individual treatment emergentadverse events (percent of patients who experienced the event at leastonce during or after study treatment) was evaluated descriptively forany noteworthy differences between test and reference treatments. AEswere also summarized by severity (mild, moderate, severe) and drugrelationship (unrelated, possibly related) for test and referencetreatments.

Disposition of Subjects

A total of 40 patients were screened for the study by the eightinvestigators. Thirty-eight of the 40 screened patients met entrancecriteria, were enrolled in the study (Table 18), and were randomized toone of the two treatments. Enrollment and randomization of the 38patients at the eight sites was as summarized in Table 18 below:

TABLE 18 ENROLLMENT AND RANDOMIZATION BY SITE AND TREATMENT 300 mgTOBI ® 420 mg TSI PARI LC PLUS ™/ PARI LC PLUS ™/ DeVilbiss InvacarePulmoAide ™ MOBILAIRE ™ Delivery System Delivery System (no. patients(no. patients Site enrolled and randomized) enrolled and randomized) 012 2 02 1 3 03 2 6 04 0 2 05 2 4 06 2 3 07 2 2 08 3 2 Total enrolled 1424 and randomized

Two of the 40 screened patients failed to meet entrance criteria andwere not enrolled in the study: one patient did not meet the protocolinclusion criterion requiring patients to have screening FEV₁% predictedresults that were ≧25%; and one patient did not meet the exclusioncriterion requiring patients to have not taken inhaled or intravenousaminoglycosides within seven days before study treatment administration.Thirty-eight patients met all study entry criteria and were randomizedto treatments. Thirty-seven of the 38 randomized patients received onedose of study treatment (Table 18). One patient was enrolled andrandomized but was withdrawn from the study before dosing due to staffinability to establish venous access for predose day 1 (visit 2) blooddraws. The 37 randomized and dosed patients constituted the intent totreat (ITT) population. All 37 patients who received study treatmentscompleted the study.

Aerosol Delivery Evaluation

Data Sets Analyzed

All 37 patients in the ITT population (i.e., those who were randomizedand received a dose of study treatment) were evaluable for the aerosoldelivery objective of the protocol. Twenty four patients received a doseof 420 mg TSI using the PARI LC PLUS™/Invacare MOBILAIRE™ DeliverySystem, and 13 patients received a dose of 300 mg TOBI® using the PARILC PLUS™/DeVilbiss PulmoAide™ Delivery System. Patient 08/137 wasexcluded from all aerosol delivery evaluations due to withdrawal fromthe study before dosing.

Demographic and Other Baseline Characteristics

Demograhic Characteristics:

Nineteen male and 18 female patients, 12 to 44 years of age anddiagnosed with cystic fibrosis, constituted the ITT population.Thirty-one patients were Caucasian, four patients were Hispanic, and twopatients were black. Gender and race distributions were similar betweenthe 420 mg TSI and 300 mg TOBI® treatment groups. On the average, ITTpatients in the 300 mg TOBI® group were approximately 2.7 years older,4.9 centimeters taller, and 10.7 kilograms heavier at screening(visit 1) than ITT patients in the 420 mg TSI group. A similar treatmentdifference in mean body weight was apparent before day 1 (visit 2)dosing, and no noteworthy change in mean weight was noted betweenscreening and day 1.

Analysis of Aerosol Delivery

Primary Aerosol Delivery Analysis: Examination of the mean plasmaconcentration-time plot for both formulations in serum (FIG. 10)indicates that tobramycin is rapidly absorbed: all subjects achievedmaximum concentrations in the time span of 10 min to 4 h. An eliminationphase was also observed in the concentration-time profiles, withindividual estimates of half-life ranging from 1.1 to 6.8 h. In sputum(FIG. 11), maximum concentrations were achieved between 15 min and 2 h,and individual estimates of half-life ranged from 0.48 to 9.47 h. Theseestimates are consistent with previous studies.

Serum and sputum pharmacokinetic parameters are summarized in Tables 19and 20 as follows.

TABLE 19 SERUM PHARMACOKINETIC PARAMETERS (MEAN ± SD) OF TOBRAMYCINAFTER ADMINISTRATION OF 300 MG TOBI AND 420 MG TSI Parameter 300 mg TOBI420 mg TSI AUC (μg h/mL) 4.38 ± 1.97 4.41 ± 1.69 C_(max) (μg/mL) 0.861 ±0.344 0.906 ± 0.542 Median t_(max) (h) 1 (1-4)* 1 (0.17-2) λ_(z) (h⁻¹)0.250 ± 0.052 0.243 ± 0.098 t_(1/2) (h) 2.78 ± 0.58 2.86 ± 1.15 CL/F(L/h) 88 ± 62 114 ± 59  V_(z)/F (L) 379 ± 325 511 ± 278

TABLE 20 SPUTUM PHARMACOKINETIC PARAMETERS (MEAN ± SD) OF TOBRAMYCINAFTER ADMINISTRATION OF 300 MG TOBI AND 420 MG TSI Parameter 300 mg TOBI420 mg TSI AUC (μg h/g) 1521 ± 845  1176 ± 686  C_(max) (μg/g) 930 ± 795 935 ± 1040 Median t_(max) (h) 0.25 (0.25-2)* 0.25 (0.25-0.25) λ_(z)(h⁻¹) 0.59 ± 0.31 0.52 ± 0.37 T_(1/2) (h) 1.17 ± 0.98 1.33 ± 0.95

The serum and sputum concentration time curves for both treatments werevirtually superimposable (FIGS. 10 and 11; Tables 19 and 20). Serumparameters (C_(max), t_(max), AUC, CL/F, Vz/F) showed no statisticallysignificant differences between the treatment groups (Table 19). Sputumparameters (AUC, C_(max), and λ_(z)) also showed no statisticallysignificant treatment differences (Table 20). Neither age nor bodyweight had a statistically significant effect on these pharmacokineticparameters. In addition, there were no statistically significantcorrelations between serum and sputum AUC, and between serum and sputumC_(max). The variability of the pharmacokinetic parameters in serum andin sputum was similar to previous trials. In summary, these findingsindicate that it is possible to achieve comparable serum and sputumlevels of tobramycin to the 300 mg TOBI® formulation by using the 420 mgTSI formulation.

Secondary Aerosol Delivery Analyses

Nebulization Time: Nebulization time was substantially reduced duringadministration of the test 420 mg TSI formulation below that observedduring administration of the marketed 300 mg TOBI® formulation. Mean±SDtotal nebulization time was 9.7±3.0 minutes during 420 mg TSIadministration compared to 18.1±3.6 minutes during 300 mg TOBI®administration (Table 21). These findings indicate that the reducednebulization times used in the 420 mg TSI treatment did not change thepharmacokinetics of tobramycin relative to the marketed 300 mg TOBI®formulation.

TABLE 21 MEAN (SD) NEBULIZATION TIME 300 mg TOBI 420 mg TSI PARI LCPLUS^(a) PARI LC PLUS^(b) Parameter PulmoAide Compressor MOBILAIRECompressor [mean (SD)] (n = 13) (n = 24) Nebulization 18.1 (3.6) 9.7(3.0) Time (min) No. pts with data 12 23 Source: Table 14.2.2.1. Notes:^(a)Reference treatment = TOBI 300 mg delivered by PARI LC PLUSnebulizer with PulmoAide compressor. Nebulization time for patient07/132 indeterminate due to interruption in dosing and unrecordedstop/start times. ^(b)Test treatment = TSI 420 mg delivered by PARI LCPLUS nebulizer with MOBILAIRE compressor. Nebulization time for patient07/126 indeterminate due to interruption in dosing and unrecordedstop/start times.

Nebulizer Weight: Nebulizer weight changes from before to after dosingindicated that the test 420 mg TSI formulation delivered less product topatients than the marketed 300, mg TOBI® formulation. Mean±SD amounts ofproduct delivered to patients was 1.86±0.53 gm during 420 mg TSIadministration and 2.74±1.64 gm during 300 mg TOBI® administration(Table 14.2.2.2), as summarized in Table 11.4-4 below. These findingslikely reflect the smaller 3.5 mL volume of TSI formulation in thenebulizer compared to the 5 mL volume of the TOBI® formulation.

TABLE 22 MEAN (SD) NEBULIZER WEIGHT AND CHANGE IN WEIGHT 300 mg TOBI^(a)PARI LC PLUS 420 mg TSI^(b) PulmoAide PARI LC PLUS Parameter CompressorMOBILAIRE Compressor [mean (SD)] (n = 13) (n = 24) Nebulizer Weight (gm)Predose 68.25 (7.30) 69.17 (0.61) No. patients with data 13 24 Postdose65.51 (6.89) 67.30 (0.80) No. patients with data 13 23 Change in weight−2.74 (1.64)^(c) −1.86 (0.53) No. patients with data 13 23 Notes:^(a)Reference treatment = TOBI 300 mg delivered by PARI LC PLUSnebulizer with PulmoAide compressor. ^(b)Test treatment = TSI 420 mgdelivered by PARI LC PLUS nebulizer with MOBILAIRE compressor. Nebulizerweight for patient 02/116 not recorded after dosing. ^(c)Theposttreatment nebulizer weight for patient 07/132 included the weight ofthe filter, and the pretreatment to posttreatment change in nebulizerweight was an increase by 2.20 gm. Excluding this erroneous value yieldsa mean (SD) change of −3.16 ( ) gm.Discussion

Aerosol delivery findings indicate that it is possible to achievecomparable serum and sputum levels of tobramycin to the 300 mg TOBI®formulation by using the 420 mg TSI formulation. Present findings alsoindicate that the reduced nebulization times and reduced amount ofproduct delivered to patients during administration of the 420 mg TSItreatment did not change the pharmacokinetics of tobramycin relative tothe marketed 300 mg TOBI® formulation.

Mean serum tobramycin concentration-time plots for both formulationsindicate that tobramycin is rapidly absorbed: all subjects achievedmaximum concentrations in the time span of 10 min to 4 h. An eliminationphase was also observed in the concentration-time profiles, withindividual estimates of half-life ranging from 1.1 to 6.8 h. In sputum,maximum concentrations were achieved between 15 min and 2 h, andindividual estimates of half-life ranged from 0.48 to 9.47 h.

The serum and sputum concentration-time curves for both treatments inthe present study were virtually superimposable. Serum parameters(C_(max), t_(max), AUC, CL/F, Vz/F) showed no statistically significantdifferences between the treatment groups. Mean (±SD) serum C_(max)results for both the 420 mg TSI and the 300 mg TOBI® groups (0.906±0.542μg/mL vs. 0.861±0.344 μg/mL, respectively) were consistent with resultsfrom previous studies.^(5,40,41) The average serum concentration oftobramycin one hour after inhalation of a single 300 mg dose of TOBI® byCF patients was 0.95 μg/mL.⁵ After 20 weeks of therapy on the TOBI®regimen, the average serum tobramycin concentration one hour afterdosing was 1.05 μg/mL.

Sputum parameters (AUC, C_(max), and λ_(z)) also showed no statisticallysignificant treatment differences in the present study. Mean (±SD)sputum C_(max) results for both the 420 mg TSI and the 300 mg TOBI®groups (935±1040 μg/g vs. 930±795 μg/g, respectively) were consistentwith results from previous studies.^(5,40,41) Sputum results in thepresent study were highly variable. By comparison, high variability oftobramycin concentration in sputum was also observed in both Phase 3trials.^(29,30) Ten minutes after inhalation of the first 300 mg dose ofTOBI® in the Phase 3 trials, the average concentration of tobramycin insputum was 1237 μg/g (ranging from 35 to 7414 μg/g). Tobramycin does notaccumulate in sputum; after 20 weeks of therapy with the TOBI® regimen,the average concentration of tobramycin at ten minutes after inhalationwas 1154 μg/g (ranging from 39 to 8085 μg/g). Two hours afterinhalation, sputum concentrations declined to approximately 14% of thetobramycin levels measured at ten minutes after inhalation.

Neither age nor body weight had a statistically significant effect onserum and sputum pharmacokinetic parameters. In addition, there were nostatistically significant correlations between serum and sputum AUC andbetween serum and sputum C_(max).

Nebulization time for the test 420 mg TSI formulation was substantiallyreduced below that observed during administration of the marketed 300 mgTOBI® formulation (mean±SD=9.7±3.0 min vs. 18.1±3.6 min, respectively).Nebulization times for the marketed 300 mg TOBI® formulation wereconsistent with previous studies.^(40,41) Therefore, the study achieveda key benchmark with the demonstration that the alternative deliverysystem, consisting of 3.5 mL of a 120 mg/mL (total 420 mg tobramycin)Tobramycin Solution for Inhalation (TSI) delivered using a PARI LC PLUS™jet nebulizer driven by an Invacare MOBILAIRE™ compressor, reducednebulization time below 10 minutes on the average.

Finally, present findings indicate that the reduced nebulization timesduring administration of the 420 mg TSI treatment did not change thepharmacokinetics of tobramycin relative to the marketed 300 mg TOBI®formulation.

Safety findings indicate that both a single dose of the 420 mg TSIformulation and a single dose of the marketed 300 mg TOBI® formulationwere well-tolerated by patients with cystic fibrosis. The incidence ofbronchospasm (≧10% relative decrease in FEV₁) was approximately 8% foreach treatment (two 420 mg TSI and one 300 mg TOBI® patients); a singlepatient in the 300 mg TOBI® group had clinically significantbronchospasm (≧10% relative decrease in FEV₁). The treatment meanrelative decrease in FEV₁ was −3.36±5.47% for 420 mg TSI and −2.14±9.62%for 300 mg TOBI®.

By comparison, in the Phase III trials of TOBI®, the median change inFEV₁ 30 minutes after the first dose of study drug had been administeredwas −1.8% in the tobramycin group. At Week 20, the median change in FEV₁was −2.0% in the tobramycin group. Because up to 95% of CF patients havebronchodilator-responsive airflow obstruction, and the within-subjectvariability for pulmonary function tests in CF patients has beendocumented to be greater than in normal patients, a ≧20% decrease inFEV₁ was considered clinically significant.³³ Twelve of 258 TOBI®patients (4.7%) had a ≧20% decrease in FEV₁ with TOBI® administration.Only two of these patients documented acute symptoms, and no patientshad a ≧20% decrease in FEV₁ more than once with TOBI®.

The present study also showed that the incidence of othertreatment-related adverse events was very low (2 of 24 TSI patients and1 of 13 TOBI® patients=8%) and did not differ between treatments. Allthree patients reported mild to moderate decreased pulmonary functiontest results, and one of the three patients also reported severe cough.Among all treatment-emergent AEs, events reported most frequently by 420mg TSI patients were cough (4 patients=17%), crepitations and sorethroat (13%), and pyrexia, nasal congestion, rhinorrhoea, and sputumincreased (8%). AEs reported most frequently by 300 mg TOBI® patientswere cough (3 patients=23%) and sore throat, dyspnoea, and rhinorrhoea(15%). These events were mostly mild to moderate in intensity (twoinstances of severe cough), were most likely related to patientsunderlying cystic fibrosis and other medical conditions, and wereconsistent with previous large Phase 3 study results.^(29,30) A singlepatient experienced serious non-drug-related symptoms (SAEs) indicativeof an exacerbation of CF. None of the patients in the study werewithdrawn due to AEs, and no other clinically significant findings werenoted in physical examinations, vital signs, or other safetymeasurements that represented an increase in risk to patients by reasonof administration of study treatments.

Conclusions

The findings of the present study indicate that it is possible toachieve comparable serum and sputum levels of tobramycin to the 300 mgTOBI® formulation by using 420 mg TSI formulation. Current findings alsoindicate that the reduced nebulization times used in the 420 mg TSItreatment did not change the pharmacokinetics of tobramycin relative tothe marketed 300 mg TOBI® formulation. Mean plasma concentration-timeplots for both formulations in serum indicate that tobramycin is rapidlyabsorbed: all subjects achieved maximum concentrations in the time spanof 10 min to 4 h. An elimination phase was also observed in theconcentration-time profiles, with individual estimates of half-liferanging from 1.1 to 6.8 h. In sputum, maximum concentrations wereachieved between 15 min and 2 h, and individual estimates of half-liferanged from 0.48 to 9.47 h. These estimates are consistent with previousstudies.

The serum and sputum concentration-time curves for both treatments werevirtually superimposable. Serum parameters (C_(max), t_(max), AUC, CL/F,Vz/F) showed no statistically significant differences between thetreatment groups. Sputum parameters (AUC, C_(max), and λ_(z)) alsoshowed no statistically significant treatment differences. Neither agenor body weight had a statistically significant effect on thesepharmacokinetic parameters. In addition, there were no statisticallysignificant correlations between serum and sputum AUC, and between serumand sputum C_(max).

During administration of the test 420 mg TSI formulation, nebulizationtime was substantially reduced below that observed during administrationof the marketed 300 mg TOBI® formulation (mean±SD=9.7±3.0 min vs.18.1±3.6 min, respectively). The apparent treatment difference in changein nebulizer weight likely reflected the different starting volumes ofTSI and TOBI® formulations in the nebulizer (mean±SD=1.86±0.53 g vs.2.74±1.64 g, respectively).

Aerosol delivery findings indicate that it is possible to achievecomparable serum and sputum levels of tobramycin to the 300 mg TOBI®formulation by using the 420 mg TSI formulation. Current findings alsoindicate that the reduced nebulization times during administration ofthe 420 mg TSI treatment did not change the pharmacokinetics oftobramycin relative to the marketed 300 mg TOBI® formulation.

While the preferred embodiments of the invention have been illustratedand described, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1. A method of treatment of a patient having an endobronchial infectioncomprising administering to the patient for inhalation a nebulized unitdose of 4.0 ml or less of an aqueous solution comprising from about 60to about 200 mg/ml of tobramycin in a physiologically acceptable carrierfor a duration of nebulization less than about 10 minutes, using aninhalation device having a rate of aerosol output of not less than about4 μl/sec, that releases at least about 75% of the loaded dose, and thatproduces aerosol particles having particle sizes between about 1 μm toabout 5 μm.
 2. A method of claim 1 wherein the unit dose comprises lessthan about 3.75 ml. of the aqueous solution.
 3. A method of claim 1wherein the unit dose comprises 3.75 ml. or less of the aqueoussolution.
 4. A method of claim 1 wherein the aqueous solution comprisesfrom about 80 to about 180 mg./ml. of tobramycin.
 5. A method of claim 1wherein the aqueous solution comprises from about 90 to about 150mg./ml. of tobramycin.
 6. A method of claim 1 wherein the unit dosecomprises 3.75 ml. or less of an aqueous solution comprising from about80 to about 180 mg./ml. of tobramycin.
 7. A method of claim 1 whereinthe duration of nebulization is less than about 8 minutes.
 8. A methodof claim 1 wherein the duration of nebulization is less than about 6minutes.
 9. A method of claim 1 wherein the inhalation device is breathactuated.
 10. A method of claim 1 wherein the inhalation device has arate of aerosol output of not less than about 5 μl/sec.
 11. A method ofclaim 1 wherein the inhalation device has a rate of aerosol output ofnot less than about 8 μl/sec.
 12. A method of claim 1 wherein theinhalation device releases at least about 80 percent of the loaded dose.13. A method of claim 1 wherein the inhalation device releases at leastabout 85 percent of the loaded dose.
 14. A method of the treatment of apatient having an endobronchial infection comprising administering tothe patient for inhalation a nebulized unit dose of 3.5 ml or less of anaqueous solution comprising from about 90 to 150 mg/ml of tobramycin ina physiologically acceptable carrier for a duration of nebulization lessthan about 6 minutes, using an inhalation device having a rate ofaerosol output of not less than about 4 μl/sec, that releases at leastabout 75% of the loaded dose, and that produces aerosol particles havinga particle size of about 1 μm to about 5 μm.